Anti-il-6 antibodies, compositions, methods and uses

ABSTRACT

The present invention relates to at least one novel chimeric, humanized or CDR-grafted anti-IL-6 antibodies derived from the murine CLB-8 antibody, including isolated nucleic acids that encode at least one such anti-IL-6 antibody, vectors, host cells, transgenic animals or plants, and methods of making and using thereof, including therapeutic compositions, methods and devices.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 13/332,973filed Dec. 21, 2011, which is a continuation of U.S. application Ser.No. 13/105,152 filed May 11, 2011, now abandoned, which is a divisionalof U.S. application Ser. No. 12/507,962 filed 23 Jul., 2009, now U.S.Pat. No. 7,955,597, which is a divisional of U.S. application Ser. No.11/832,323 filed 1 Aug. 2007, now U.S. Pat. No. 7,612,182 issued 3 Nov.2009, which is a divisional of U.S. application Ser. No. 10/280,716,filed 26 Oct. 2002, now U.S. Pat. No. 7,291,721 issued 6 Nov. 2007,which claims the benefit of U.S. Provisional Application Ser. No.60/332,743 filed 14 Nov. 2001. The entire contents of the aforementionedapplications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antibodies, including specifiedportions or variants, specific for at least one Interleukin-6 (IL-6 alsoknown as Interferon β2) protein or fragment thereof, as well as nucleicacids encoding such anti-IL-6 antibodies, complementary nucleic acids,vectors, host cells, and methods of making and using thereof, includingtherapeutic formulations, administration and devices.

2. Related Art

Interleukin-6 (IL-6) is a pro-inflammatory cytokine that is produced bymany different cell types. In vivo, stimulated monocytes, fibroblasts,and endothelial cells represent the main sources of IL-6. Other cellssuch as macrophages, T and B lymphocytes, granulocytes, keratinocytes,mast cells, osteoblasts, chrondrocytes, glial cells, and smooth musclecells also produce IL-6 after stimulation (Kishimoto, T., Blood 74:1-10(1989) and Kurihara, N. et al., J. Immunology 144:4226-4230 (1990)).Several tumor cells also produce IL-6 (Smith, P. C. et al. Cytokine andGrowth Factor Reviews 12:33-40 (2001)) and recently IL-6 was indicatedto be a prognostic factor for prostate cancer progression (Nakashima, J.et al. Clinical Cancer Research 6:2702-2706 (2000)). IL-6 production canbe regulated by IL-6 itself and depending upon cell type, IL-6 canstimulate or inhibit its own synthesis.

IL-6 can bind to the IL-6 receptor expressed on mitogen-activated Bcells, T cells, peripheral monocytes, and certain tumors (Ishimi, Y. etal., J. Immunology 145:3297-3303 (1990)). The IL-6 receptor has at leasttwo different components and is composed of an alpha chain called gp80that is responsible for IL-6 binding and a beta chain designated gp130that is needed for signal transduction (Adebanjo, O. et al., J. CellBiology 142:1347-1356 (1998) and Poli, V. et al., EMBO 13:1189-1196(1994)). The cytokine family which includes IL-6, LIF, Oncostatin M,IL-11, CNTF, and CT-1 all signal through gp130 after binding to theircognate receptors. In addition, all members of the IL-6 cytokine familycan induce hepatic expression of acute phase proteins (Bellido, T. etal., J. Clin. Investigation 97:431-437 (1996)). 87908790.

There are at least two major biological functions of IL-6: mediation ofacute phase proteins and acting as a differentiation and activationfactor (Avvisti, G. et al., Baillieres Clinical Hematology 8:815-829(1995) and Poli, V. et al., EMBO 13:1189-1196 (1994)). Acute phaseproteins are known to regulate immune responses, mediate inflammation,and play a role in tissue remodeling. As a differentiation andactivation factor, IL-6 induces B cells to differentiate and secreteantibody, it induces T cells to differentiate into cytotoxic T cells,activates cell signaling factors, and promotes hematopoiesis (Ishimi, Y.et al., J Immunology 145:3297-3303 (1990)). IL-6 is prominently involvedin many critical bodily functions and processes. As a result,physiological processes including bone metabolism, neoplastictransformation, and immune and inflammatory responses can be enhanced,suppressed, or prevented by manipulation of the biological activity ofIL-6 in vivo by means of an antibody (Adebanjo, O. et al., J. CellBiology 142:1347-1356 (1998)).

Although IL-6 is involved in many pathways, IL-6 knockout mice have anormal phenotype, they are viable and fertile, and these animals showslightly decreased number of T cells and decreased acute phase proteinresponse to tissue injury (Kopf M et al., Impaired immune andacute-phase responses in interleukin-6-deficient mice, Nature;368(6469):339-42, 1994). In contrast, transgenic mice that over-expressIL-6 develop neurologic disease such as neurodegeneration, astrocytosis,cerebral vasculogenesis, and these mice do not develop a blood brainbarrier (Campbell et al., Neurologic Disease Induced in Transgenic Miceby Cerebral Overexpression of Interleukin 6 PNAS 90: 10061-10065. 1993).

Recent studies have indicated that a Mab to IL-6 can inhibit in vivogrowth of prostate tumors (Smith, P. C. and Keller, E. T., The Prostatein press and Okamoto, M. et al., Cancer Research 57:141-146 (1997) andrenal carcinoma (Weissglas, M. et al., The Journal of Urology153:554-557 (1995)). In addition to a direct effect on tumor growth,blocking IL-6 production can also chemo-sensitize and enhance cytotoxicefficacy (Smith, P. C. et al. Cytokine and Growth Factor Reviews12:33-40 (2001)). Collectively, literature teaches us that blocking IL-6activity can inhibit bone degradation, tumor growth and cancer cachexia.

Passive immunotherapy employing non-human, polyclonal (e.g., anti-sera)or monoclonal antibodies (Mabs) and fragments thereof (e.g., proteolyticdigestion products thereof) are potential therapeutic agents that arebeing developed as treatments for various diseases. However, antibodiescomposed of non-human portions are known to elicit an immune responsewhen administered to humans. This immune response makes repeatedantibody administration often unsuitable for therapy and may result inan immune complex mediated clearance of the antibodies from circulation,thus reducing the therapeutic benefit to the patients. Examples ofconditions that may be attributed to repeat administration of antibodiescomposed of non-human portions are serum sickness and anaphylaxis.

In an attempt to avoid these and other problems, a number of approachesincluding chimerization and “humanization” have been pursued to reducethe immunogenicity of the antibodies/fragments thereof. These approacheshave produced antibodies having reduced immunogenicity. These antibodiesare substantially of human origin, with only the complementarydetermining regions (CDR's) and certain framework residue that influenceCDR conformation being of non human original. Novel human or humanizedmonoclonal antibodies are therefore particularly useful alone or incombination with existing molecules for immunotherapeutic uses.

Accordingly, there is a need to provide a high affinity, neutralizingchimeric or human antibodies to IL-6 or fragments thereof that overcomeone more of these problems, as well as improvements over knownantibodies or fragments thereof for use in preventing, treating,ameliorating, or diagnosing conditions related to the IL-6.

Murine monocolonal antibodies to IL-6 produced from a hybridoma cellline are known for example in U.S. Pat. No. 5,618,700. U.S. Pat. No.5,856,135 discloses reshaped human antibodies to human IL-6 drived froma mouse monoclonal antibody SK2 in which the complementary determiningregions (CDR's) from the variable region of the mouse antibody SK2 aretransplanted into the variable region of a human antibody and joined tothe constant region of a human antibody.

Other murine monoclonal antibodies have been described and categorizedas neutralizing, that is preventing receptor binding, ornon-neutralizing (Brakenhoff et al, J. Immunol. (1990) (145:561). Amongthis set of antibodies, neutralizing monoclonal antibodies to IL-6 canbe divided into two groups; and the putative epitopes on the IL-6molecule designated Site I and Site II. Site I prevent binding to thegp80 (IL6R) and therefore prevent gp130 activation. The Site I epitopewas further characterized as comprising regions of both amino terminaland carboxy terminal portions of the IL-6 molecule. Site II bindersprevent gp130 activation and therefore may recognize a conformationalepitope involved in signalling.

A murine IL-6 monoclonal antibody referred to as CLB-6/8 or CLB-8, whichhas high affinity for IL-6, binds to the Site I epitope, is known(Brakenhoff et al supra), but the antigen binding domains (CDR regions)of this antibody are not known. As described above, however, the murineantibody is highly immunogenic in humans and its therapeutic value istherefore limited. There is thus a continuing need for antibodies toIL-6 that exhibit high affinity and a favorable pharmaceutical profile.

SUMMARY OF THE INVENTION

The present invention provides isolated chimeric, humanized and/orCDR-grafted anti-IL-6 antibodies, having at least one antigen bindingregion derived from the high affinity CLB-8 anti-IL-6 antibody, as wellas anti-IL-6 antibody compositions, encoding or complementary nucleicacids, vectors, host cells, compositions, formulations, devices,transgenic animals, transgenic plants related thereto, and methods ofmaking and using thereof, as described and enabled herein, incombination with what is known in the art. The antibody of the inventionspecifically neutralizes human IL-6 with high affinity.

The present invention provides at least one isolated human-mousechimeric, humanized or CDR-grafted anti-IL-6 CLB-8 antibody (“cCLB-8antibody”) as described herein. The cCLB-8 antibody according to thepresent invention includes any protein or peptide molecule thatcomprises at least one complementarity determining region (CDR) of aheavy or light chain or a ligand binding portion thereof, derived fromthe murine CLB-8 monoclonal antibody, in combination with a heavy chainor light chain constant region, a framework region, or any portionthereof, that can be incorporated into an antibody of the presentinvention. In one embodiment the invention is directed to an anti-IL-6chimeric antibody comprising two light chains and two heavy chains, eachof the chains comprising at least part of a human constant region and atleast part of a variable region (v) derived from the murine CLB8monoclonal antibody having specificity to human IL-6, said antibodybinding with high affinity to an inhibiting and/or neutralizing epitopeof human IL-6, such as the antibody cCLB-8. The invention also includesfragments or a derivative of such an antibody, such as one or moreportions of the antibody chain, such as the heavy chain constant,joining, diversity or variable regions, or the light chain constant,joining or variable regions.

The antibody can comprise at least one specified portion of at least onecomplementarity determining region (CDR) (e.g., CDR1, CDR2 or CDR3 ofthe heavy or light chain variable region) derived from the murine CLB-8monoclonal antibody, and/or at least one constant or variable frameworkregion or any portion thereof. The antibody amino acid sequence canfurther optionally comprise at least one specified substitution,insertion or deletion as described herein or as known in the art.

Preferred antibodies of the present invention include those chimeric,humanized and/or CDR grafted antibodies that will competitively inhibitin vivo binding to human IL-6 of anti-IL-6 murine CLB-8, chimericanti-IL-6 CLB-8, or an antibody having substantially the same bindingcharacteristics, as well as fragments and regions thereof.

Preferred antibodies of the present invention are those that bindepitopes recognized by CLB-8 and cCLB-8, which are included in the SiteIepitope as described by Brackenhoff et al. (supra). Preferred methodsfor determining monoclonal antibody specificity and affinity bycompetitive inhibition can be found in Harlow, et al, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988), hereby incorporated by reference into the presentapplication. At least one antibody of the invention binds at least onespecified epitope specific to human IL-6 protein, subunit, fragment,portion or any combination thereof, to which the CLB-8 monoclonalantibody binds. The epitope can comprise at least one antibody bindingregion to which the CLB-8 antibody binds, which epitope is preferablycomprised of at least 1-5 amino acids of at least one portion thereof,such as but not limited to, at least one functional, extracellular,soluble, hydrophillic, external or cytoplasmic domain of human IL-6protein, or any portion thereof.

In one aspect, the present invention provides at least one isolatedmammalian anti-IL-6 cCLB-8 antibody, comprising at least one variableregion comprising SEQ ID NO:7 or 8 and the nucleic acid sequencesencoding them (SEQ ID NO: 15 or 16).

In another aspect, the present invention provides at least one isolatedmammalian anti-IL-6 cCLB-8 antibody, comprising either (i) all of theheavy chain complementarity determining regions (CDR) amino acidsequences of SEQ ID NOS:1, 2, and 3 and the nucleic acid sequencesencoding them (SEQ ID NOS: 9-11); or (ii) all of the light chain CDRamino acids sequences of SEQ ID NOS:4, 5, and 6 and the nucleic acidsequences encoding them (SEQ ID NOS: 12-14).

In another aspect, the present invention provides at least one isolatedmammalian anti-IL-6 cCLB-8 antibody, comprising at least one heavy chainor light chain CDR having the amino acid sequence of at least one of SEQID NOS: 1, 2, 3, 4, 5, or 6 and the nucleic acid sequences encoding them(SEQ ID NOS: 9-14).

In other aspect the present invention provides at least one isolatedmammalian chimeric, humanized or CDR-grafted anti-IL-6 cCLB-8 antibody,comprising at least one human CDR, wherein the antibody specificallybinds at least one epitope comprising at least 1-3 amino acids of theepitope of human IL-6 to which the CLB-8 antibody binds.

The at least one antibody can optionally further bind IL-6 with anaffinity (K_(d)) of at least 10⁻⁹ M, preferably at least 10⁻¹⁰ M, and/orsubstantially neutralize at least one activity of at least one IL-6protein. In a preferred embodiment, the antibody binds IL-6 with anaffinity (K_(d)) of at least 1×10⁻¹¹M, preferably 5×10⁻¹¹ neutralizeshuman IL-6.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding the aforementioned specific anti-IL-6 antibodies, comprising atleast one specified sequence, domain, portion or variant thereof. Thepresent invention further provides recombinant vectors comprising saidanti-IL-6 antibody nucleic acid molecules, host cells containing suchnucleic acids and/or recombinant vectors, as well as methods of makingand/or using such antibody nucleic acids, vectors and/or host cells.Thus, the invention comprises an isolated nucleic acid encoding at leastone isolated mammalian anti-IL-6 cCLB-8 antibody; an isolated nucleicacid vector comprising the isolated nucleic acid, and/or a prokaryoticor eukaryotic host cell comprising the isolated nucleic acid. The hostcell can optionally be at least one selected from COS-1, COS-7, HEK293,BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphomacells, or any derivative, immortalized or transformed cell thereof. Alsoprovided is a method for producing at least one anti-IL-6 cCLB-8antibody, comprising translating the antibody encoding nucleic acidunder conditions in vitro, in vivo or in situ, such that the IL-6antibody is expressed in detectable or recoverable amounts.

The present invention further provides at least one IL-6 anti-idiotypeantibody to at least one cCLB-8 anti-IL-6 antibody of the presentinvention. The anti-idiotype antibody includes any protein or peptidecontaining molecule that comprises at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that can be incorporated into ananti-idiotype antibody to the antibody of the present invention. Ananti-idiotype antibody of the invention can include or be derived fromany mammal, such as but not limited to a human, a mouse, a rabbit, arodent, a primate, and the like.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding at least one IL-6 anti-idiotype antibody, comprising at leastone specified sequence, domain, portion or variant thereof. The presentinvention further provides recombinant vectors comprising said IL-6anti-idiotype antibody encoding nucleic acid molecules, host cellscontaining such nucleic acids and/or recombinant vectors, as well asmethods of making and/or using such anti-idiotype antibody nucleicacids, vectors and/or host cells.

The present invention also provides at least one method for expressingat least one aforementioned anti-IL-6 antibody, or IL-6 anti-idiotypeantibody in a host cell, comprising culturing a host cell as describedherein under conditions wherein at least one anti-IL-6 antibody isexpressed in detectable and/or recoverable amounts.

Also provided is a method for producing at least one isolated anti-IL-6antibody of the present invention, comprising providing a transgenicanimal or transgenic plant or plant cell capable of expressing theantibody in recoverable amounts. Further provided in the presentinvention is at least one anti-IL-6 antibody produced by the abovemethod.

The present invention also provides at least one composition comprising(a) an isolated anti-IL-6 cCLB-8 antibody encoding nucleic acid and/orantibody as described herein; and (b) a suitable carrier or diluent. Thecarrier or diluent can optionally be pharmaceutically acceptable,according to known carriers or diluents. The composition can optionallyfurther comprise at least one further compound, protein or composition.

The present invention further provides at least one anti-IL-6 cCLB-8antibody method or composition, for administering a therapeuticallyeffective amount to modulate or treat at least one IL-6 relatedcondition in a cell, tissue, organ, animal or patient and/or, prior to,subsequent to, or during a related condition, as known in the art and/oras described herein. Thus, the invention provides a method fordiagnosing or treating an IL-6 related condition in a cell, tissue,organ or animal, comprising contacting or administering a compositioncomprising an effective amount of at least one isolated anti-IL-6 cCLB-8antibody of the invention with, or to, the cell, tissue, organ oranimal. The method can optionally further comprise using an effectiveamount of 0.001-50 mg/kilogram of the cells, tissue, organ or animal.The method can optionally further comprise using the contacting or theadministrating by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.The method can optionally further comprise administering, prior,concurrently, or after the antibody contacting or administering at leastone composition comprising an effective amount of at least one compoundor protein selected from at least one of a detectable label or reporter,a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromsucula-r blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steriod, an epinephrineor analog thereof, a cytotoxic or other anti-cancer agent, ananti-metabolite such as methotrexate, an anti-proliferative agent, acytokine, or a cytokine antagonist.

The present invention further provides at least one anti-IL-6 cCLB-8antibody method for diagnosing at least one IL-6 related condition in acell, tissue, organ, animal or patient and/or, prior to, subsequent to,or during a related condition, as known in the art and/or as describedherein.

The present invention also provides at least one composition, deviceand/or method of delivery for diagnosing of at least one anti-IL-6antibody, according to the present invention.

Also provided is a composition comprising at least one isolatedchimeric, human or humanized anti-IL-6 cCLB-8 antibody and at least onepharmaceutically acceptable carrier or diluent. The composition canoptionally further comprise an effective amount of at least one compoundor protein selected from at least one of a detectable label or reporter,a cytotoxic or other anti-cancer agent, an anti-metabolite such asmethotrexate, an anti-proliferative agent, a cytokine, or a cytokineantagonist, a TNF antagonist, an antirheumatic, a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NTHE), an analgesic, ananesthetic, a sedative, a local anethetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog.

Also provided is a medical device, comprising at least one isolatedmammalian anti-IL-6 antibody of the invention, wherein the device issuitable to contacting or administering the at least one anti-IL-6antibody by at least one mode selected from parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

Also provided is an article of manufacture for human pharmaceutical ordiagnostic use, comprising packaging material and a container comprisinga solution or a lyophilized form of at least one isolated mammaliananti-IL-6 antibody of the present invention. The article of manufacturecan optionally comprise having the container as a component of aparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal delivery device or system.

The present invention further provides any invention described herein.

DESCRIPTION OF THE FIGURES

FIG. 1: Graph showing binding of cCLB8 to human recombinant IL-6.

FIG. 2: Graph showing the inhibition of IL-6 mediated IgM mu secretionfrom SKW6.4 cells by cCLB8.

FIG. 3: Graph showing the inhibition of IL-6 mediated MCP-1 productionby cCLB8.

FIG. 4: Image of a western blot showing cCLB8 inhibition of IL-6signaling in THP-1 human monocytic leukemia cells.

FIG. 5: Graph showing the inhibition by cCLB8 of IL-6 induced serumamyloid A production from HepG2 cells.

FIG. 6: Graph showing the ability of cCLB8 to neutralize rhIL-6-inducedcell proliferation.

FIG. 7: Graph showing the relative reduction in host body weight loss inhuman tumor bearing mice treated with both anti-human and anti-mouseIL-6 antibodies.

FIG. 8A-G: Graph showing the serum inhibition study profiles of 7anti-idiotype antibodies.

FIG. 9: Graph showing the inhibition of cCLB8 binding to human IL-6 byanti-id Mabs.

FIG. 10: Graph showing the anti-id binding to cCLB-8 pre-bound to humanIL-6.

DETAILED DESCRIPTION OF THE INVENTION Citations

All publications or patents cited herein are entirely incorporatedherein by reference as they show the state of the art at the time of thepresent invention and/or to provide description and enablement of thepresent invention. Publications refer to any scientific or patentpublications, or any other information available in any media format,including all recorded, electronic or printed formats. The followingreferences are entirely incorporated herein by reference: Ausubel, etal., ed., Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y. (1989);Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor,N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology,John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., CurrentProtocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

Amino Acid Codes

The amino acids that make up anti-IL-6 antibodies of the presentinvention are often abbreviated. The amino acid designations can beindicated by designating the amino acid by its single letter code, itsthree letter code, name, or three nucleotide codon(s) as is wellunderstood in the art (see Alberts, B., et al., Molecular Biology of TheCell, Third Ed., Garland Publishing, Inc., New York, 1994):

DEFINITIONS

As used herein, an “anti-Interleukin-6 cCLB-8 antibody,” “anti-IL-6cCLB-8 antibody,” “anti-IL-6 cCLB-8 antibody portion,” or “anti-IL-6cCLB-8 antibody fragment” and/or “anti-IL-6 cCLB-8 antibody variant” andthe like include any protein or peptide containing molecule thatcomprises at least a portion of an immunoglobulin molecule, containingat least one complementarity determining region (CDR) of a heavy orlight chain or a ligand binding portion thereof derived from the murineCLB-8 monoclonal antibody in combination with a heavy chain or lightchain variable region, a heavy chain or light chain constant region, aframework region, or any portion thereof, of non-murine origin,preferably of human origen, which can be incorporated into an antibodyof the present invention. Such antibody is capable of modulating,decreasing, antagonizes, mitigates, aleviates, blocks, inhibits,abrogates and/or interferes with at least one IL-6 activity or binding,or with IL-6 receptor activity or binding, in vitro, in situ and/or invivo. As a non-limiting example, a suitable anti-IL-6 antibody,specified portion or variant of the present invention can bind with highaffinity to an inhibiting and/or neutralizing epitope of human IL-6recognized by the CLB-8 monoclonal antibody. A suitable anti-IL-6antibody, specified portion, or variant can also optionally affect atleast one of IL-6 activity or function, such as but not limited to, RNA,DNA or protein synthesis, IL-6 release, IL-6 receptor signaling,membrane IL-6 cleavage, IL-6 activity, IL-6 production and/or synthesis.

The term “antibody” is further intended to encompass antibodies,digestion fragments, specified portions and variants thereof, includingantibody mimetics or comprising portions of antibodies that mimic thestructure and/or function of an antibody or specified fragment orportion thereof, including single chain antibodies and fragmentsthereof; each containing at least one CDR derived from the CLB-8monoclonal antibody. Functional fragments include antigen-bindingfragments that bind to a mammalian IL-6. For example, antibody fragmentscapable of binding to IL-6 or portions thereof, including, but notlimited to Fab (e.g., by papain digestion), Fab′ (e.g., by pepsindigestion and partial reduction) and F(ab′)₂ (e.g., by pepsindigestion), facb (e.g., by plasmin digestion), pFc′ (e.g., by pepsin orplasmin digestion), Fd (e.g., by pepsin digestion, partial reduction andreaggregation), Fv or scFv (e.g., by molecular biology techniques)fragments, are encompassed by the invention (see, e.g., Colligan,Immunology, supra).

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the CH₁ domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

As used herein “chimeric” antibodies or “humanized” antibodies or“CDR-grafted” include any combination of the herein described murineCDR's with one or more proteins or peptides derived from a non-murine,preferably, human antibody. In accordance with the invention, chimericor humanized antibodies are provided wherein the CDR's are derived fromthe murine CLB-8 antibody capable of binding human IL-6 and at least aportion, or the remainder of the antibody is derived from one or morehuman antibodies. Thus, the human part of the antibody may include theframework, C_(L), C_(H) domains (e.g., C_(H)1, C_(H)2, C_(H)3), hinge,(V_(L), V_(H))) regions which are substantially non-immunogenic inhumans. The regions of the antibody that are derived from humanantibodies need not have 100% identity with human antibodies. In apreferred embodiment, as many of the human amino acid residues aspossible are retained in order for the immunogenicity to be negligible,but the human residues may be modified as necessary to support theantigen binding site formed by the CDR's while simultaneously maximizingthe humanization of the antibody. Such changes or variations optionallyand preferably retain or reduce the immunogenicity in humans or otherspecies relative to non-modified antibodies. It is pointed out that ahumanized antibody can be produced by a non-human animal or prokaryoticor eukaryotic cell that is capable of expressing functionally rearrangedhuman immunoglobulin (e.g., heavy chain and/or light chain) genes.Further, when the antibody is a single chain antibody, it can comprise alinker peptide that is not found in native human antibodies. Forexample, an Fv can comprise a linker peptide, such as two to about eightglycine or other amino acid residues, which connects the variable regionof the heavy chain and the variable region of the light chain. Suchlinker peptides are considered to be of human origin.

Antibodies of the Present Invention

In accordance with the present invention, the anti-IL-6 cCLB-8 antibodycomprises an antibody in which the variable region or CDRs are derivedfrom the murine CLB-8 antibody capable of binding to and inhibiting thefunction of human IL-6 and the framework and constant regions of theantibody are derived from one or more human antibodies. The variableregion or CDRs derived from the murine CLB-8 antibody preferably havefrom about 90% to about 100% identity with the variable region or CDRsof the murine CLB-8 antibody, although any and all modifications,including substitutions, insertions and deletions, are contemplated solong as the chimeric antibody maintains the ability to bind to andinhibit IL-6. The regions of the chimeric, humanized or CDR-graftedantibodies that are derived from human antibodies need not have 100%identity with the human antibodies. In a preferred embodiment, as manyof the human amino acid residues as possible are retained in order thanimmunogenicity is negligible, but the human residues, in particularresidues of the framework region, are substituted as required and astaught hereinbelow in accordance with the present invention. Suchmodifications as disclosed herein are necessary to support the antigenbinding site formed by the CDRs while simultaneously maximizing thehumanization of the antibody.

The CLB-8 murine monoclonal antibody against human IL-6 is known in theart (Brakenhoff et al, supra), but the CDR regions of this antibody havenot heretofore been disclosed. The present invention, for the firsttime, discloses chimeric, humanized or CDR grafted antibodies derivedfrom the CDR regions of the CLB-8 murine monoclonal antibody and methodsfor preparing such antibodies. In accordance with the present invention,the cDNA (SEQ ID NO: 15) and amino acid sequences of the heavy chain(SEQ ID NO: 7) of murine CLB-8 heavy chain is provided at Example 2. ThecDNA and deduced amino acid sequence of the murine CLB-8 light chain(SEQ ID NO. 8) is also provided in Example 2 (SEQ ID NO: 16). Each ofthe heavy and light chain variable regions contain three CDRs thatcombine to form the antigen binding site. The three CDRs are surroundedby four FR regions that primarily function to support the CDRs. Thesequences of the CDRs within the sequences of the variable regions ofthe heavy and light chains can be identified by computer-assistedalignment according to Kabat et al. (1987) in Sequences of Proteins ofImmunological Interest, 4^(th) ed., United States Department of Healthand Human Services, U.S. Government Printing Office, Washington, D.C.,or by molecular modeling of the variable regions, for example utilizingthe ENCAD program as described by Levitt (1983) J. Mol. Biol. 168:595.

In a preferred embodiment the CDRs are derived from murine monoclonalantibody CLB-8. The preferred heavy chain CDRs have the followingsequences:

CDR1 (SEQ ID NO: 1) SFAMS CDR2 (SEQ ID NO: 2) EISSGGSYTYYPDTVTG CDR3(SEQ ID NO: 3) GLWGYYALDY

The preferred light chain CDRs have the following sequences:

CDR1 (SEQ. ID NO: 4) SASSSVSYMY CDR2 (SEQ. ID NO: 5) DTSNLAS CDR3(SEQ. ID NO: 6) QQWSGYPYT

The sequences of the CDRs of the murine CLB-8 antibody, may be modifiedby insertions, substitutions and deletions to the extent that theCDR-grafted antibody maintains the ability to bind to and inhibit humanIl-6. The ordinarily skilled artisan can ascertain the maintenance ofthis activity by performing the functional assays described hereinbelow.The CDRs can have, for example, from about 50% to about 100% homology tothe CDRs of SEQ ID NOS: 1-6. In a preferred embodiment the CDRs havefrom about 80% to about 100% homology to the CDRs of SEQ ID NOS: 1-6. Ina more preferred embodiment the CDRs have from about 90% to about 100%homology to the CDRs of SEQ ID NOS: 1-6. In a most preferred embodimentthe CDRs have from about 100% homology to the CDRs of SEQ ID NOS:1-6.

Alternatively, the entire heavy chain variable region and light chainvariable region of the murine CLB-8 antibody as set forth in Example 2(SEQ.ID NOS. 7 and 8) may be combined with the human constant andframework regions to form the chimeric cCLB-8 antibody of the presentinvention.

Human genes which encode the constant (C) regions of the chimericantibodies, fragments and regions of the present invention can bederived from a human fetal liver library, by known methods. Human Cregion genes can be derived from any human cell including those whichexpress and produce human immunoglobulins. The human C_(H) region can bederived from any of the known classes or isotypes of human H chains,including gamma, μ, α, δ, ε, and subtypes thereof, such as G1, G2, G3and G4. Since the H chain isotype is responsible for the variouseffector functions of an antibody, the choice of C_(H) region will beguided by the desired effector functions, such as complement fixation,or activity in antibody-dependent cellular cytotoxicity (ADCC).Preferably, the C_(H) region is derived from gamma 1 (IgG1).

The human C_(L) region can be derived from either human L chain isotype,kappa or lambda, preferably kappa.

Genes encoding human immunoglobulin C regions are obtained from humancells by standard cloning techniques (Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1989) and Ausubel et al, eds. CurrentProtocols in Molecular Biology (1987-1993)). Human C region genes arereadily available from known clones containing genes representing thetwo classes of L chains, the five classes of H chains and subclassesthereof. Chimeric antibody fragments, such as F(ab¹)₂ and Fab, can beprepared by designing a chimeric H chain gene which is appropriatelytruncated. For example, a chimeric gene encoding an H chain portion ofan F(ab¹)₂ fragment would include DNA sequences encoding the CH1 domainand hinge region of the H chain, followed by a translational stop codonto yield the truncated molecule.

Generally, in one example, chimeric antibodies, fragments and regions ofthe present invention are produced by cloning DNA segments encoding theH and L chain antigen-binding regions of the CLB-8 anti IL-6 specificantibody, and joining these DNA segments to DNA segments encoding C_(H)and C_(L) regions, respectively, to produce chimericimmunoglobulin-encoding genes.

Thus, in a preferred embodiment, a fused chimeric gene is created whichcomprises a first DNA segment that encodes at least the antigen-bindingregion of non-human origin, such as a functionally rearranged V regionwith joining (J) segment, linked to a second DNA segment encoding atleast a part of a human C region.

The sequences of the variable regions of the murine CLB-8 antibody, maybe modified by insertions, substitutions and deletions to the extentthat the chimeric antibody maintains the ability to bind to and inhibithuman IL-6. The ordinarily skilled artisan can ascertain the maintenanceof this activity by performing the functional assays describedhereinbelow. The variable regions can have, for example, from about 50%to about 100% homology to the variable regions of SEQ ID NOS:7-8. In apreferred embodiment the variable regions have from about 80% to about100% homology to the variable regions of SEQ ID NOS: 7-8. In a morepreferred embodiment the variable regions have from about 90% to about100% homology to the variable regions of SEQ ID NOS: 7-8. In a mostpreferred embodiment the variable regions have from about 100% homologyto the CDRs of SEQ ID NOS: 1-6.

For convenience, the numbering scheme of Kabat et al., has been adoptedherein. Residues are designated by lower case numbers or hyphens asnecessary to conform the present sequences to the standard Kabatnumbered sequence.

In accordance with the present invention, in the case of a CDR-graftedor humanized antibody where the CDR region of the CLB-8 antibody iscombined with a human region, residues may be retained in the FR regionwhich are idiosyncratic to the parent antibody, e.g. CLB-8. Residuesthat have been demonstrated to be critical in the humanization of otherantibodies may also be retained. The foregoing guidelines are followedto the extent necessary to support the antigen binding site formed bythe CDRs while simultaneously maximizing the humanization of theantibody.

The amino acid sequence of a representative heavy chain variable regionderived from murine monoclonal antibody CLB-8 and a human antibody areshown in Example 2 below.

The amino acid sequence of a representative chimeric light chainvariable region derived from murine monoclonal antibody CLB-8 and ahuman antibody is also shown in Example 2.

A chimeric antibody containing variable regions from the murine CLB-8antibody has been demonstrated in accordance with the present inventionto be as effective as murine monoclonal antibody CLB-8 in binding toIL-6.

Methods for engineering or humanizing non-human or human antibodies canbe used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcewhich is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence. Known human Ig sequences are disclosed, e.g.,www._ncbi.nlm.nih.gov/entrez/query.fcgi; www._atcc.org/phage/hdb.html;www._sciquest.com/; www._abcam.com/;www._antibodyresource.com/onlinecomp.html;www._public.iastate.edu/˜pedro/research_tools.html;www._mgen.uni-heidelberg.de/SD/IT/IT.html;www._whfreeman.com/immunology/CH05/kuby05.htm;www._library.thinkquest.org/12429/Immune/Antibody.html;www._hhmi.org/grants/lectures/1996/vlab/;www._path.cam.ac.uk/˜mrc7/mikeimages.html; www._antibodyresource.com/;mcb.harvard.edu/BioLinks/Immunology.html. www._immunologylink.com/;pathbox.wustl.edu/˜hcenter/index.html; www._biotech.ufl.edu/˜hcl/;www._pebio.com/pa/340913/340913.html;www._nal.usda.gov/awic/pubs/antibody/;www._m.ehime-u.ac.jp/˜yasuhito/Elisa.html; www._biodesign.com/table.asp;www.icnet.uk/axp/facs/davies/links.html;www._biotech.ufl.edu/˜fccl/protocol.html;www._isac-net.org/sites_geo.html;aximt1.imt.uni-marburg.de/˜rek/AEPStart.html;baserv.uci.kun.nl/˜jraats/links1.html;www._recab.uni-hd.de/immuno.bme.nwu.edu/;www._mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;www._ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;www._biochem.ucl.ac.uk/˜martin/abs/index.html; antibody.bath.ac.uk/;abgen.cvm.tamu.edu/lab/wwwabgen.html;www._unizh.ch/˜honegger/AHOseminar/Slide01.html;www._cryst.bbk.ac.uk/˜ubcg17s/;www._nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www._path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html;www._ibt.unam.mx/vir/structure/stataim.html;www._biosci.missouri.edu/smithgp/index.html;www._cryst.bioc.cam.ac.uk/˜fmolina/Web-pages/Pept/spottech.html;www._jerini.de/fr_products.htm; www._patents. ibm.com/ibm.html.Kabat etal. Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. Generally part or all of the non-human or human CDRsequences are maintained while the non-human sequences of the variableand constant regions are replaced with human or other amino acids.Antibodies can also optionally be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding. Humanization or engineering of antibodiesof the present invention can be performed using any known method, suchas but not limited to those described in, Winter (Jones et al., Nature321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen etal., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296(1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al.,Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993), U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514,5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352,6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539;4,816,567, PCT/: US98/16280, US96/18978, US91/09630, US91/05939,US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424,WO90/14430, EP 229246, each entirely incorporated herein by reference,included references cited therein

The human constant region of the chimeric antibody of the invention canbe of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and cancomprise a kappa or lambda light chain. In one embodiment, the humanconstant region comprises an IgG heavy chain or defined fragment, forexample, at least one of isotypes, IgG1, IgG2, IgG3 or IgG4. In anotherembodiment, the anti-human IL-6 human antibody comprises an IgG1 heavychain and a IgG1 K light chain. The isolated anti-IL-6 antibodies of thepresent invention comprise antibody amino acid sequences disclosedherein encoded by any suitable polynucleotide as well as. Preferably,the antibody or antigen-binding fragment binds human IL-6 and, therebypartially or substantially neutralizes at least one biological activityof the protein. The cCLB-8 antibody, or specified portion or variantthereof, partially or preferably substantially neutralizes at least onebiological activity of at least one IL-6 protein or fragment and therebyinhibit activities mediated through the binding of IL-6 to the IL-6receptor or through other IL-6-dependent or mediated mechanisms. As usedherein, the term “neutralizing antibody” refers to an antibody that caninhibit an IL-6-dependent activity by about 20-120%, preferably by atleast about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay. Thecapacity of an anti-IL-6 antibody to inhibit an IL-6-dependent activityis preferably assessed by at least one suitable IL-6 protein or receptorassay, as described herein and/or as known in the art.

At least one antibody of the invention binds at least one specifiedepitope specific to at least one IL-6 protein, subunit, fragment,portion or any combination thereof to which the CLB-8 antibody binds.The at least one epitope can comprise at least one antibody bindingregion that comprises at least one portion of the protein, which epitopeis preferably comprised of at least one extracellular, soluble,hydrophillic, external or cytoplasmic portion of the protein. Generally,the human antibody or antigen-binding fragment of the present inventionwill comprise an antigen-binding region that comprises at least onehuman complementarity determining region (CDR1, CDR2 and CDR3) of SEQ IDNOS. 1, 2 and 3 or variant of at least one heavy chain variable regionand at least one human complementarity determining region (CDR4, CDR5and CDR6) (SEQ ID NO. 4, 5 and 6) or variant of at least one light chainvariable region. As a non-limiting example, the antibody orantigen-binding portion or variant can comprise at least one of theheavy chain CDR3 having the amino acid sequence of SEQ ID NO:3, and/or alight chain CDR3 having the amino acid sequence of SEQ ID NO:6. In aparticular embodiment, the antibody or antigen-binding fragment can havean antigen-binding region that comprises at least a portion of at leastone heavy chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acidsequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS:1, 2,and/or 3). In another particular embodiment, the antibody orantigen-binding portion or variant can have an antigen-binding regionthat comprises at least a portion of at least one light chain CDR (i.e.,CDR4, CDR5 and/or CDR6) having the amino acid sequence of thecorresponding CDRs 4, 5 and/or 6 (e.g., SEQ ID NOS: 4, 5, and/or 6). Ina preferred embodiment the three heavy chain CDRs and the three lightchain CDRs of the antibody or antigen-binding fragment have the aminoacid sequence of the corresponding CDR of at least one of mAb cCLB8,Chimeric anti-IL-6 Mab, as described herein. Such antibodies can beprepared by chemically joining together the various portions (e.g.,CDRs, framework) of the antibody using conventional techniques, bypreparing and expressing a (i.e., one or more) nucleic acid moleculethat encodes the antibody using conventional techniques of recombinantDNA technology or by using any other suitable method and using any ofthe possible redundant codons that will result in expression of apolypeptide of the invention, for example, SEQ ID NO: 15 or 16.

Antibodies that bind to human IL-6 and that comprise the defined heavyor light chain variable region or CDR regions can be prepared usingsuitable methods, such as phage display (Katsube, Y., et al., Int J Mol.Med, 1(5):863-868 (1998)) or methods that employ transgenic animals, asknown in the art and/or as described herein. For example, the antibody,specified portion or variant can be expressed using the encoding nucleicacid or portion thereof in a suitable host cell.

As stated, the invention also relates to antibodies, antigen-bindingfragments, immunoglobulin chains and CDRs comprising amino acids in asequence that is substantially the same as an amino acid sequencedescribed herein. Such anti-IL-6 antibodies can include one or moreamino acid substitutions, delations or additions, either from naturalmutations or human manipulation, as specified herein. Preferably, suchantibodies or antigen-binding fragments and antibodies comprising suchchains or CDRs can bind human IL-6 with high affinity (e.g., K_(D) lessthan or equal to about 10⁻⁹ M). Amino acid sequences that aresubstantially the same as the sequences described herein includesequences comprising conservative amino acid substitutions, as well asamino acid deletions and/or insertions. A conservative amino acidsubstitution refers to the replacement of a first amino acid by a secondamino acid that has chemical and/or physical properties (e.g, charge,structure, polarity, hydrophobicity/hydrophilicity) that are similar tothose of the first amino acid. Conservative substitutions includereplacement of one amino acid by another within the following groups:lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate(E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine(Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine(I), proline (P), phenylalanine (F), tryptophan (W), methionine (M),cysteine (C) and glycine (G); F, W and Y; C, S and T.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for any given anti-IL-6 antibody, fragment or variant willnot be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, asspecified herein.

Amino acids in an anti-IL-6 antibody of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g.,Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science244:1081-1085 (1989)). The latter procedure introduces single alaninemutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least one IL-6 neutralizing activity. Sites that arecritical for antibody binding can also be identified by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992)and de Vos, et al., Science 255:306-312 (1992)).

Anti-IL-6 antibodies of the present invention can include, but are notlimited to, at least one portion, sequence or combination selected from5 to all of the contiguous amino acids of at least one of SEQ ID NOS:1,2, 3, 4, 5, 6.

A(n) anti-IL-6 antibody can further optionally comprise a polypeptide ofat least one of 70-100% of the contiguous amino acids of at least one ofSEQ ID NOS:7, 8.

In one embodiment, the amino acid sequence of an immunoglobulin chain,or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of thecorresponding chain of at least one of SEQ ID NOS:7, 8. For example, theamino acid sequence of a light chain variable region can be comparedwith the sequence of SEQ ID NO:8, or the amino acid sequence of a heavychain CDR3 can be compared with SEQ ID NO:7. Preferably, 70-100% aminoacid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or anyrange or value therein) is determined using a suitable computeralgorithm, as known in the art.

Exemplary heavy chain and light chain variable regions sequences areprovided in SEQ ID NOS: 7, 8. The antibodies of the present invention,or specified variants thereof, can comprise any number of contiguousamino acid residues from an antibody of the present invention, whereinthat number is selected from the group of integers consisting of from10-100% of the number of contiguous residues in an anti-IL-6 antibody.Optionally, this subsequence of contiguous amino acids is at least about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids inlength, or any range or value therein. Further, the number of suchsubsequences can be any integer selected from the group consisting offrom 1 to 20, such as at least 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-1000% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

The modified antibodies and antigen-binding fragments of the inventioncan comprise one or more organic moieties that are covalently bonded,directly or indirectly, to the antibody.

Each organic moiety that is bonded to an antibody or antigen-bindingfragment of the invention can independently be a hydrophilic polymericgroup, a fatty acid group or a fatty acid ester group. As used herein,the term “fatty acid” encompasses mono-carboxylic acids anddi-carboxylic acids. A “hydrophilic polymeric group,” as the term isused herein, refers to an organic polymer that is more soluble in waterthan in octane. For example, polylysine is more soluble in water than inoctane. Thus, an antibody modified by the covalent attachment ofpolylysine is encompassed by the invention. Hydrophilic polymerssuitable for modifying antibodies of the invention can be linear orbranched and include, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodies ofthe invention include, for example, n-dodecanoate (C₁₂, laurate),n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈, stearate),n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂, behenate),n-triacontanoate (C₃₀₋), n-tetracontanoate (C₄₀₋), cis-Δ9-octadecanoate(C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate (C₂₀, arachidonate),octanedioic acid, tetradecanedioic acid, octadecanedioic acid,docosanedioic acid, and the like. Suitable fatty acid esters includemono-esters of dicarboxylic acids that comprise a linear or branchedlower alkyl group. The lower alkyl group can comprise from one to abouttwelve, preferably one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis(Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996);Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and themethods described in Hermanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

The antibodies of the invention can bind human IL-6 with a wide range ofaffinities (K_(D)). In a preferred embodiment, at least one human mAb ofthe present invention can optionally bind human IL-6 with high affinity.For example, a mAb can bind human IL-6 with a K_(D) equal to or lessthan about 10⁻⁷ M, such as but not limited to, 0.1-9.9 (or any range orvalue therein) ×10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10¹², 10⁻¹³ or any rangeor value therein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(a)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

Anti-IL-6 cCLB-8 antibodies useful in the methods and compositions ofthe present invention are characterized by high affinity binding to IL-6and optionally and preferably having low toxicity. In particular, anantibody, specified fragment or variant of the invention, where theindividual components, such as the variable region, constant region andframework, individually and/or collectively, optionally and preferablypossess low immunogenicity, is useful in the present invention. Theantibodies that can be used in the invention are optionallycharacterized by their ability to treat patients for extended periodswith measurable alleviation of symptoms and low and/or acceptabletoxicity. Low or acceptable immunogenicity and/or high affinity, as wellas other suitable properties, can contribute to the therapeutic resultsachieved. “Low immunogenicity” is defined herein as raising significantHAHA, HACA or HAMA responses in less than about 75%, or preferably lessthan about 50% of the patients treated and/or raising low titres in thepatient treated (less than about 300, preferably less than about 100measured with a double antigen enzyme immunoassay) (Elliott et al.,Lancet 344:1125-1127 (1994), entirely incorporated herein by reference).

When cCLB8 is compared to other IL-6-specific antibodies CLB.IL-6/14 andCLB.IL-6/16, one can see the distinct characteristics of antibodyaffinity and epitope specificity. cCLB8, an antibody that binds IL-6 andnormally blocks the interaction between IL-6 and its receptor, caninhibit nearly 100% of IL-6 function as illustrated in both the IL-6dependent 7TD1 cell proliferation bioassay and the IL-6 binding to IL-6receptor Luminex based assay. In contrast, CLB.IL-6/16, an antibody thatbinds IL-6, but neutralizes by sterically hindering the interactionbetween the IL-6/IL-6R complex and the gp130 signaling component, caninhibit only 62% of the bound biotin-IL-6. Finally, an antibody thatbinds IL-6 but does not interfere with its biological activity, as inCLB.IL-6/14, displays no inhibition of biotin-IL-6 binding the solidphase sIL-6R/gp80.

Bispecific, heterospecific, heteroconjugate or similar antibodies canalso be used that are monoclonal, humanized, antibodies that havebinding specificities for at least two different antigens. In thepresent case, one of the binding specificities is for at least one IL-6protein, the other one is for any other antigen. Methods for makingbispecific antibodies are known in the art. Traditionally, therecombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two heavy chains have different specificities (Milstein and Cuello,Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453,6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985,5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549,4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986),each entirely incorporated herein by reference.

Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencesencoding at least 70-100% of the contiguous amino acids of at least oneof SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, specified fragments, variants orconsensus sequences thereof, or a deposited vector comprising at leastone of these sequences, a nucleic acid molecule of the present inventionencoding at least one cCLB-8 anti-IL-6 antibody can be obtained usingmethods described herein or as known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain (e.g., SEQ ID NOS:1-3) or light chain (e.g.,SEQ ID NOS: 4-6); nucleic acid molecules comprising the coding sequencefor an anti-IL-6 antibody or variable region (e.g., SEQ ID NOS:15 or16); and nucleic acid molecules which comprise a nucleotide sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode at least one anti-IL-6antibody as described herein and/or as known in the art. Of course, thegenetic code is well known in the art. Thus, it would be routine for oneskilled in the art to generate such degenerate nucleic acid variantsthat code for specific anti-IL-6 antibodies of the present invention.See, e.g., Ausubel, et al., supra, and such nucleic acid variants areincluded in the present invention. Non-limiting examples of isolatednucleic acid molecules of the present invention include SEQ ID NOS:9-16; corresponding to non-limiting examples of a nucleic acid encoding,respectively, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3, HCvariable region and LC variable region.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-IL-6 antibody caninclude, but are not limited to, those encoding the amino acid sequenceof an antibody fragment, by itself; the coding sequence for the entireantibody or a portion thereof; the coding sequence for an antibody,fragment or portion, as well as additional sequences, such as the codingsequence of at least one signal leader or fusion peptide, with orwithout the aforementioned additional coding sequences, such as at leastone intron, together with additional, non-coding sequences, includingbut not limited to, non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences that play a role in transcription,mRNA processing, including splicing and polyadenylation signals (forexample—ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding anantibody can be fused to a marker sequence, such as a sequence encodinga peptide that facilitates purification of the fused antibody comprisingan antibody fragment or portion.

Polynucleotides Which Selectively Hybridize to a Polynucleotide asDescribed Herein

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides of the present inventioncan be used to identify, isolate, or amplify partial or full-lengthclones in a deposited library. In some embodiments, the polynucleotidesare genomic or cDNA sequences isolated, or otherwise complementary to, acDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably at least 85% or 90% full-length sequences, andmore preferably at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of an antibody encoded by the polynucleotides described herein.The polynucleotides of this invention embrace nucleic acid sequencesthat can be employed for selective hybridization to a polynucleotideencoding an antibody of the present invention. See, e.g., Ausubel,supra; Colligan, supra, each entirely incorporated herein by reference.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra)

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor, etal; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson,et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 toGyllensten, et al; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat.No. 4,994,370 to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S.Pat. No. 4,656,134 to Ringold) and RNA mediated amplification that usesanti-sense RNA to the target sequence as a template for double-strandedDNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with thetradename NASBA), the entire contents of which references areincorporated herein by reference. (See, e.g., Ausubel, supra; orSambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can beused to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example a cDNA or a genomicsequence encoding an antibody of the present invention, can be used toconstruct a recombinant expression cassette that can be introduced intoat least one desired host cell. A recombinant expression cassette willtypically comprise a polynucleotide of the present invention operablylinked to transcriptional initiation regulatory sequences that willdirect the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in intron) of a non-heterologous formof a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

Vectors and Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one anti-IL-6 antibody by recombinant techniques, as is wellknown in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al.,supra, each entirely incorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one antibody of the present invention can be expressed in amodified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of an antibody to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage.

Also, peptide moieties can be added to an antibody of the presentinvention to facilitate purification. Such regions can be removed priorto final preparation of an antibody or at least one fragment thereof.Such methods are described in many standard laboratory manuals, such asSambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra,Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention.

Alternatively, nucleic acids of the present invention can be expressedin a host cell by turning on (by manipulation) in a host cell thatcontains endogenous DNA encoding an antibody of the present invention.Such methods are well known in the art, e.g., as described in U.S. Pat.Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirelyincorporated herein by reference.

Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653,SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readilyavailable from, for example, American Type Culture Collection, Manassas,Va. (www.atcc.org). Preferred host cells include cells of lymphoidorigin such as myeloma and lymphoma cells. Particularly preferred hostcells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) andSP2/0-Ag14 cells (ATCC Accession Number CRL-1851). In a particularlypreferred embodiment, the recombinant cell is a P3X63Ab8.653 or aSP2/0-Ag14 cell.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Production of an Antibody

At least one anti-IL-6 antibody of the present invention can beoptionally produced by a cell line, a mixed cell line, an immortalizedcell or clonal population of immortalized cells, as well known in theart. See, e.g., Ausubel, et al., ed., Current Protocols in MolecularBiology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor, N.Y. (1989); Harlow and Lane, antibodies, a Laboratory Manual,Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., CurrentProtocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001);Colligan et al., Current Protocols in Protein Science, John Wiley &Sons, NY, N.Y., (1997-2001), each entirely incorporated herein byreference.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or thelike, or heteromylomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart. See, e.g., www.atcc.org, www.lifetech.com., and the like, withantibody producing cells, such as, but not limited to, isolated orcloned spleen, peripheral blood, lymph, tonsil, or other immune or Bcell containing cells, or any other cells expressing heavy or lightchain constant or variable or framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference.

Any other suitable host cell can also be used for expressingheterologous or endogenous nucleic acid encoding an antibody, specifiedfragment or variant thereof, of the present invention. The fused cells(hybridomas) or recombinant cells can be isolated using selectiveculture conditions or other suitable known methods, and cloned bylimiting dilution or cell sorting, or other known methods. Cells whichproduce antibodies with the desired specificity can be selected by asuitable assay (e.g., ELISA).

Antibodies of the present invention can also be prepared using at leastone anti-IL-6 antibody encoding nucleic acid to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. Such animals can be providedusing known methods. See, e.g., but not limited to, U.S. Pat. Nos.5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616, 5,565,362;5,304,489, and the like, each of which is entirely incorporated hereinby reference.

Antibodies of the present invention can additionally be prepared usingat least one anti-IL-6 antibody encoding nucleic acid to providetransgenic plants and cultured plant cells (e.g., but not limited totobacco and maize) that produce such antibodies, specified portions orvariants in the plant parts or in cells cultured therefrom. As anon-limiting example, transgenic tobacco leaves expressing recombinantproteins have been successfully used to provide large amounts ofrecombinant proteins, e.g., using an inducible promoter. See, e.g.,Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencecited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to know methods. See also,e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October,1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., PlantPhysiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans.22:940-944 (1994); and references cited therein. See, also generally forplant expression of antibodies, but not limited to, Each of the abovereferences is entirely incorporated herein by reference.

Purification of an Antibody

An anti-IL-6 antibody can be recovered and purified from recombinantcell cultures by well-known methods including, but not limited to,protein A purification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

Cloning and Expression of IL-6 Antibody in Mammalian Cells

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, theantibody coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pIRESlneo, pRetro-Off,pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL andPMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of IL-6 antibody. Plasmid pC4is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). Theplasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (e.g., alpha minus MEM, LifeTechnologies, Gaithersburg, Md.) supplemented with the chemotherapeuticagent methotrexate. The amplification of the DHFR genes in cellsresistant to methotrexate (MTX) has been well documented (see, e.g., F.W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C.Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M.A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasingconcentrations of MTX develop resistance to the drug by overproducingthe target enzyme, DHFR, as a result of amplification of the DHFR gene.If a second gene is linked to the DHFR gene, it is usually co-amplifiedand over-expressed. It is known in the art that this approach can beused to develop cell lines carrying more than 1,000 copies of theamplified gene(s). Subsequently, when the methotrexate is withdrawn,cell lines are obtained that contain the amplified gene integrated intoone or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human b-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express the IL-6in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc.Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For the polyadenylation ofthe mRNA other signals, e.g., from the human growth hormone or globingenes can be used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete IL-6 antibody is used, e.g., aspresented in SEQ ID NOS: 7, and 8, corresponding to HC and LC variableregions of a IL-6 antibody of the present invention, according to knownmethod steps. Isolated nucleic acid encoding a suitable human constantregion (i.e., HC and LC regions) is also used in this construct.

The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary (CHO) cells lacking an active DHFR gene are usedfor transfection. 5 mg of the expression plasmid pC4 is cotransfectedwith 0.5 mg of the plasmid pSV2-neo using lipofectin. The plasmidpSV2neo contains a dominant selectable marker, the neo gene from Tn5encoding an enzyme that confers resistance to a group of antibioticsincluding G418. The cells are seeded in alpha minus MEM supplementedwith 1 μg/ml G418. After 2 days, the cells are trypsinized and seeded inhybridoma cloning plates (Greiner, Germany) in alpha minus MEMsupplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 μg/ml G418.After about 10-14 days single clones are trypsinized and then seeded in6-well petri dishes or 10 ml flasks using different concentrations ofmethotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing atthe highest concentrations of methotrexate are then transferred to new6-well plates containing even higher concentrations of methotrexate (1mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated untilclones are obtained that grow at a concentration of 100-200 mM.Expression of the desired gene product is analyzed, for instance, bySDS-PAGE and Western blot or by reverse phase HPLC analysis.

Anti-Idiotype Antibodies to Anti-Il-6 Antibody Composition

In addition to monoclonal or chimeric anti-IL-6 antibodies, the presentinvention is also directed to an anti-idiotypic (anti-Id) antibodyspecific for such antibodies of the invention. An anti-Id antibody is anantibody which recognizes unique determinants generally associated withthe antigen-binding region of another antibody. The anti-Id can beprepared by immunizing an animal of the same species and genetic type(e.g. mouse strain) as the source of the Id antibody with the antibodyor a CDR containing region thereof. The immunized animal will recognizeand respond to the idiotypic determinants of the immunizing antibody andproduce an anti-Id antibody. The anti-Id antibody may also be used as an“immunogen” to induce an immune response in yet another animal,producing a so-called anti-anti-Id antibody.

Anti-Il-6 Antibody Compositions

The present invention also provides at least one anti-IL-6 antibodycomposition comprising at least one, at least two, at least three, atleast four, at least five, at least six or more anti-IL-6 antibodiesthereof, as described herein and/or as known in the art that areprovided in a non-naturally occurring composition, mixture or form. Suchcompositions comprise non-naturally occurring compositions comprising atleast one or two full length, C- and/or N-terminally deleted variants,domains, fragments, or specified variants, of the anti-IL-6 antibodyamino acid sequence selected from the group consisting of 70-100% of thecontiguous amino acids of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, orspecified fragments, domains or variants thereof. Preferred anti-IL-6antibody compositions include at least one or two full length,fragments, domains or variants as at least one CDR or LBR containingportions of the anti-IL-6 antibody sequence of 70-100% of SEQ ID NOS:1,2, 3, 4, 5, 6, or specified fragments, domains or variants thereof.Further preferred compositions comprise 40-99% of at least one of70-100% of SEQ ID NOS:1, 2, 3, 4, 5, 6, or specified fragments, domainsor variants thereof. Such composition percentages are by weight, volume,concentration, molarity, or molality as liquid or dry solutions,mixtures, suspension, emulsions or colloids, as known in the art or asdescribed herein.

Anti-IL-6 antibody compositions of the present invention can furthercomprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising at least oneanti-IL-6 antibody to a cell, tissue, organ, animal or patient in needof such modulation, treatment or therapy, optionally further comprisingat least one selected from at least one TNF antagonist (e.g., but notlimited to a TNF antibody or fragment, a soluble TNF receptor orfragment, fusion proteins thereof, or a small molecule TNF antagonist),an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose,azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquinesulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial(e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a fluororquinolone, a macrolide, apenicillin, a sulfonamide, a tetracycline, another antimicrobial), anantipsoriatic, a corticosteriod, an anabolic steroid, a diabetes relatedagent, a mineral, a nutritional, a thyroid agent, a vitamin, a calciumrelated hormone, an antidiarrheal, an antitussive, an antiemetic, anantiulcer, a laxative, an anticoagulant, an erythropieitin (e.g.,epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim(GM-CSF, Leukine), an immunization, an immunoglobulin, animmunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), agrowth hormone, a hormone replacement drug, an estrogen receptormodulator, a mydriatic, a cycloplegic, an alkylating agent, anantimetabolite, a mitotic inhibitor, a radiopharmaceutical, anantidepressant, antimanic agent, an antipsychotic, an anxiolytic, ahypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthmamedication, a beta agonist, an inhaled steroid, a leukotriene inhibitor,a methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha(Pulmozyme), a cytokine or a cytokine antagonist. Non-limiting examplesof such cytokines include, but are not limited to, any of IL-1 to IL-23.Suitable dosages are well known in the art. See, e.g., Wells et al.,eds., Pharmacotherapy Handbook, 2^(nd) Edition, Appleton and Lange,Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000),each of which references are entirely incorporated herein by reference.

Such anti-cancer or anti-infectives can also include toxin moleculesthat are associated, bound, co-formulated or co-administered with atleast one antibody of the present invention. The toxin can optionallyact to selectively kill the pathologic cell or tissue. The pathologiccell can be a cancer or other cell. Such toxins can be, but are notlimited to, purified or recombinant toxin or toxin fragment comprisingat least one functional cytotoxic domain of toxin, e.g., selected fromat least one of ricin, diphtheria toxin, a venom toxin, or a bacterialtoxin. The term toxin also includes both endotoxins and exotoxinsproduced by any naturally occurring, mutant or recombinant bacteria orviruses which may cause any pathological condition in humans and othermammals, including toxin shock, which can result in death. Such toxinsmay include, but are not limited to, enterotoxigenic E. coli heat-labileenterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin,Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-1),Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcalenterotoxins and the like. Such bacteria include, but are not limitedto, strains of a species of enterotoxigenic E. coli (ETEC),enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7),Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcuspyogenes), Shigella species (e.g., Shigella dysenteriae, Shigellaflexneri, Shigella boydii, and Shigella sonnei), Salmonella species(e.g., Salmonella typhi, Salmonella cholera-suis, Salmonellaenteritidis), Clostridium species (e.g., Clostridium perfringens,Clostridium dificile, Clostridium botulinum), Camphlobacter species(e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacter species,(e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria,Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides,Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae, Vibriosparahemolyticus), Klebsiella species, Pseudomonas aeruginosa, andStreptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds.,Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp239-254, Plenum Medical Book Co., New York (1991); Mandell et al,Principles and Practice of Infectious Diseases, 3d. Ed., ChurchillLivingstone, N.Y. (1990); Berkow et al, eds., The Merck Manual, 16thedition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack et al, Science,248:705-711 (1990), the contents of which references are incorporatedentirely herein by reference.

Anti-IL-6 antibody compounds, compositions or combinations of thepresent invention can further comprise at least one of any suitableauxiliary, such as, but not limited to, diluent, binder, stabilizer,buffers, salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the anti-IL-6 antibody, fragment or variantcomposition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Anti-IL-6 antibody compositions can also include a buffer or a pHadjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid saltssuch as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts such as citrate.

Additionally, anti-IL-6 antibody compositions of the invention caninclude polymeric excipients/additives such as polyvinylpyrrolidones,ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the anti-IL-6 antibody, portion or variantcompositions according to the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed.,Williams & Williams, (1995), and in the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), the disclosuresof which are entirely incorporated herein by reference. Preferredcarrier or excipient materials are carbohydrates (e.g., saccharides andalditols) and buffers (e.g., citrate) or polymeric agents.

Formulations

As noted above, the invention provides for stable formulations, which ispreferably a phosphate buffer with saline or a chosen salt, as well aspreserved solutions and formulations containing a preservative as wellas multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one anti-IL-6 antibody in apharmaceutically acceptable formulation. Preserved formulations containat least one known preservative or optionally selected from the groupconsisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concentration ormixture can be used as known in the art, such as 0.001-5%, or any rangeor value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1-2%m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol(e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g.,0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9,1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5,0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one anti-IL-6 antibody with the prescribed buffersand/or preservatives, optionally in an aqueous diluent, wherein saidpackaging material comprises a label that indicates that such solutioncan be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30,36, 40, 48, 54, 60, 66, 72 hours or greater.

The invention further comprises an article of manufacture, comprisingpackaging material, a first vial comprising lyophilized at least oneanti-IL-6 antibody, and a second vial comprising an aqueous diluent ofprescribed buffer or preservative, wherein said packaging materialcomprises a label that instructs a patient to reconstitute the at leastone anti-IL-6 antibody in the aqueous diluent to form a solution thatcan be held over a period of twenty-four hours or greater.

The at least one anti-IL-6 antibody used in accordance with the presentinvention can be produced by recombinant means, including from mammaliancell or transgenic preparations, or can be purified from otherbiological sources, as described herein or as known in the art.

The range of at least one anti-IL-6 antibody in the product of thepresent invention includes amounts yielding upon reconstitution, if in awet/dry system, concentrations from about 1.0 μg/ml to about 1000 mg/ml,although lower and higher concentrations are operable and are dependenton the intended delivery vehicle, e.g., solution formulations willdiffer from transdermal patch, pulmonary, transmucosal, or osmotic ormicro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one anti-IL-6 antibody and apreservative selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one anti-IL-6 antibody andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one anti-IL-6 antibody inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized at least one anti-IL-6antibody that is reconstituted with a second vial containing water, apreservative and/or excipients, preferably a phosphate buffer and/orsaline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2 to about 40° C. and retain the biologically activity of the proteinfor extended periods of time, thus, allowing a package label indicatingthat the solution can be held and/or used over a period of 6, 12, 18,24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used,such label can include use up to 1-12 months, one-half, one and a half,and/or two years.

The solutions of at least one anti-IL-6 antibody in the invention can beprepared by a process that comprises mixing at least one antibody in anaqueous diluent. Mixing is carried out using conventional dissolutionand mixing procedures. To prepare a suitable diluent, for example, ameasured amount of at least one antibody in water or buffer is combinedin quantities sufficient to provide the protein and optionally apreservative or buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one anti-IL-6antibody that is reconstituted with a second vial containing the aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneanti-IL-6 antibody that is reconstituted with a second vial containingthe aqueous diluent. The clear solution in this case can be up to oneliter or even larger in size, providing a large reservoir from whichsmaller portions of the at least one antibody solution can be retrievedone or multiple times for transfer into smaller vials and provided bythe pharmacy or clinic to their customers and/or patients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution such as BD Pens, BDAutojector®, Humaject® NovoPen®, B-D® Pen, AutoPen®, and OptiPen®,GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®,Biojector®, Iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®,e.g., as made or developed by Becton Dickensen (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com); NationalMedical Products, Weston Medical (Peterborough, UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,www.mediject.com). Recognized devices comprising a dual vial systeminclude those pen-injector systems for reconstituting a lyophilized drugin a cartridge for delivery of the reconstituted solution such as theHumatroPen®.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute the at least one anti-IL-6 antibody in theaqueous diluent to form a solution and to use the solution over a periodof 2-24 hours or greater for the two vial, wet/dry, product. For thesingle vial, solution product, the label indicates that such solutioncan be used over a period of 2-24 hours or greater. The presentlyclaimed products are useful for human pharmaceutical product use.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one anti-IL-6 antibody and a selectedbuffer, preferably a phosphate buffer containing saline or a chosensalt. Mixing the at least one antibody and buffer in an aqueous diluentis carried out using conventional dissolution and mixing procedures. Toprepare a suitable formulation, for example, a measured amount of atleast one antibody in water or buffer is combined with the desiredbuffering agent in water in quantities sufficient to provide the proteinand buffer at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilized atleast one anti-IL-6 antibody that is reconstituted with a second vialcontaining a preservative or buffer and excipients in an aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

At least one anti-IL-6 antibody in either the stable or preservedformulations or solutions described herein, can be administered to apatient in accordance with the present invention via a variety ofdelivery methods including SC or IM injection; transdermal, pulmonary,transmucosal, implant, osmotic pump, cartridge, micro pump, or othermeans appreciated by the skilled artisan, as well-known in the art.

Therapeutic Applications

IL-6, due to its pleiotropic activity, is implicated in the pathology ofa variety of diseases. Therefore, a high affinity, neutralizing chimericor human antibody to IL-6 would be desirable to be used in IL-6 relateddiseases such as cancer, cachexia, SLE, rheumatoid arthritis,osteoporosis, brain trauma, cerebral edema, depression, and CHF. cCLB8or any derivatives of this mAb including chimeric or humanized, orfragments can be used in alleviating bone pain, inhibiting growth oftumors such as melanoma, renal, prostate, breast, lung, colon cancer andmultiple myeloma, lymphoproliferative disorders and other diseases inwhich IL-6 has been implicated. This antibody can be used either as asingle agent or in combination with other therapeutic agents. Inaddition, this Mab can be used as a chemosensitizer whereby it canincrease therapeutic efficacy of cytotoxic agents. This antibody can beused as a radiosensitizer whereby it can improve efficacy of radiation.It can also be used in combination with other tumor-immunomodulatingagents such as IL-2, IL-12 and/or IFNalpha.

Thus, the present invention also provides a method for modulating ortreating at least one IL-6 related disease, in a cell, tissue, organ,animal, or patient, as known in the art or as described herein, using atleast one anti-IL-6 antibody of the present invention.

IL-6 is known to enhance proliferation, differentiation and survival ofmalignant plasma cells in multiple myeloma (MM) through an autocrine ora paracrine mechanism that involves the inhibition of apoptosis of themalignant cells. MM is an incurable malignant plasma cell disorderwhere, blocking IL-6 has been postulated to be an effective therapy(Anderson et al., Multiple Myeloma: New Insights and TherapeuticApproaches. Hematology: 147-165, 2000). IL-6 also has a tumorigeniceffect in basal cell carcinoma where IL-6 transfected cells showedincreased tumor growth rate by both suppressing apoptosis and activelypromoting (Jee et al., Overexpression of interleukin-6 in human basalcell carcinoma cell lines increases anti-apoptotic activity andtumorigenic potency. Oncogene, Vol. 20, No. 2 pp. 198-208, 2001). IL-6can also promote resistance of breast cancer cells to chemotherapy byinducing mdr1 gene expression (mdr1 and metallothionein pathways) (Conzeet al, Autocrine Production of Interleukin 6 Causes Multidrug Resistancein Breast Cancer Cells. Cancer Res 61: 8851-8858, 2001).

The ability of IL-6 to mediate tumor cell survival and diseaseprogression was confirmed by the inhibitory effects of an anti-IL-6 mAbon tumor growth both in vitro and in vivo. It was reported that blockadeof IL-6 can inhibit growth of human brain tumors (glioblastoma) in vitro(Goswami et al., Interleukin-6-mediated autocrine growth promotion inhuman glioblastoma multiforme cell line U87MG. J Neurochem 71:1837-1845, 1998). Using the same approach it was shown that injection ofmurine CLB8 anti-IL-6 antibody prolonged the survival of human tumorbearing mice (Mauray et al., Epstein-Barr virus-dependentlymphoproliferative disease: critical role of IL-6. Eur J Immunol;30(7):2065-73, 2000). It was also reported that mCLB8 anti-IL-6 antibodyregressed growth of human renal carcinoma tumors and decreased serumcalcium concentrations in nude mice (Weisglass et al., The role ofinterleukin-6 in the induction of hypercalcemia in renal cell carcinomatransplanted into nude mice. Endocrinology 138(5):1879-8, 1995). CLB-8antibody also regressed established human hormone refractory prostatetumor xenografts in mice (Smith et al. 2001) Anti-interleukin-6monoclonal antibody induces regression of human prostate cancerxenografts in nude mice (Smith and Keller, Prostate; 48(1):47-53).

IL-6 can also be a prognostic factor and a marker for malignancies. Inrenal cell carcinoma (RCC) high levels of IL-6 were reported tocorrelate with tumor metastasis and eventually to poor prognosis andshort survival (Jean-Yves Blay et al. 1992). Moreover, in RCC, elevatedserum IL-6 is associated with poor response to IL-2 therapy (Fumagalliet al. 1999) Pretreatment serum markers and lymphocyte response tointerleukin-2 therapy. Br J Cancer 80(3-4):407-11 and correlated withthe degree of IL-2 associated toxicity (Capuron et al. 2001) Associationbetween immune activation and early depressive symptoms in cancerpatients treated with interleukin-2-based therapy.Psychoneuroendocrinology; 26(8):797-808.

Elevated levels of IL-6 also correlated with poor prognosis and thepresence of metastatic disease in breast cancer (Kurebayashi 2000 andBenoy 2002) Regulation of interleukin-6 secretion from breast cancercells and its clinical implications. Breast Cancer; 7(2):124-9. Seruminterleukin 6, plasma VEGF, serum VEGF, and VEGF platelet load in breastcancer patients. Clin Breast Cancer; 2(4):311-5.

IL-6 is hypothesized to be a causative factor in cancer-relatedmorbidity such as asthenia/cachexia and bone resorption. Tumor-inducedcachexia (Cahlin et al. 2000) and bone resorption (subsequenthypercalcemia) (Sandhu et al. 1999) were found to be diminished in IL-6knockout mice. Cancer-associated depression, and cerebral edemasecondary to brain tumors have also been associated with high levels ofIL-6 (Musselman et al. 2001). cCLB8 antilL-6 antibody of the inventionalso inhibited human melanoma and human prostate carcinoma inducedcachexia in nude mice.

Clinical Experience with Anti-IL-6 Agents

Several clinical trials using monoclonal antibodies against IL-6 havebeen conducted in multiple diseases including plasma cell leukemia,multiple myeloma, B-lympho-proliferative disorder, rheumatoid arthritis,renal carcinoma, and AIDS associated lymphoma.

A Phase I dose escalating study with the anti-IL-6 cCLB-8 antibody ofthe present invention for the treatment of refractory patients withadvanced stage multiple myeloma (N=12) demonstrated that some patientshad disease stabilization. After discontinuation of treatment there wasacceleration in the increase of M protein levels, suggesting diseasere-bound after the withdrawal of therapy. Anti-IL-6 cCLB-8 antibodyinhibited free circulating IL-6. Most importantly no toxicity (excepttransient thrombocytopenia in two heavily pretreated patients) orallergic reactions were observed. C-reactive protein (CRP) decreasedbelow detection level in all patients. Anti-IL-6 cCLB-8 antibodydemonstrated a long circulating half-life of 17.8 days, and there was nohuman anti-chimeric antibody (HACA) immune response observed (van Zaanenet al. 1998). Administration of CNTO 328 did not cause changes in bloodpressure, pulse rate, temperature, hemoglobin, liver functions and renalfunctions. Except for transient thrombocytopenia in two heavilypretreated patients, no toxicity or allergic reactions were observed,and there was no human anti-chimeric antibody (HACA) immune responseobserved. Three patients developed infection-related complicationsduring therapy, however, a possible relation with anti-IL-6 cCLB-8antibody was unlikely because infectious complications are common in endstage multiple myeloma and are a major cause of death. In addition allthree patients were able to respond to their infection even in thepresence of anti-IL-6 cCLB-8 antibody, suggesting that anti-IL-6 therapyis not able to block IL-6 during infection. No treatment-associatedfatalities were reported. In conclusion, results from this study suggestthat anti-IL-6 cCLB-8 antibody was safe in multiple myeloma patients.

Thus, the present invention provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: multiplemyeloma, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL),B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chromicmyelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairycell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin'sdisease, a malignamt lymphoma, non-hodgkin's lymphoma, Burkitt'slymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma,renal cell carcinoma, pancreatic carcinoma, prostatic carcinoma,nasopharyngeal carcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas,sarcomas, malignant melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer related bone pain; the suppression ofcancer metastasis; the amelioration of cancer cachexia; and thetreatment of inflammatory diseases such as mesangial proliferativeglomerulonephritis and the like. Such a method can optionally be used incombination with, by administering before, concurrently or afteradministration of such IL-6 antibody, radiation therapy, ananti-angiogenic agent, a chemotherapeutic agent, a farnesyl transferaseinhibitor or the like.

The present invention also provides a method for modulating or treatingat least one Il-6 mediated immune related disease, in a cell, tissue,organ, animal, or patient including, but not limited to, at least one ofrheumatoid arthritis, juvenile rheumatoid arthritis, systemic onsetjuvenile rheumatoid arthritis, psoriatic arthritis, ankylosingspondilitis, gastric ulcer, seronegative arthropathies, asteoarthritis,inflammatory bowel disease, ulverative colitis, systemic lupuserythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/opticneuritis, idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever,perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria,systemic anaphalaxis, dermatitis, pernicious anemia, hemolyticdisesease, thrombocytopenia, graft rejection of any organ or tissue,kidney translplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, sleep apnea, obesity, heartfailure, sinusitis, inflammatory bowel disease, and the like. See, e.g.,the Merck Manual, 12th-17th Editions, Merck & Company, Rahway, N.J.(1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells etal., eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998,2000), each entirely incorporated by reference.

The present invention also provides a method for modulating or treatingat least one infectious disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: acute orchronic bacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis (A, B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, e. coli0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, vital-associated hemaphagocytic syndrome, vitalencephalitis/aseptic meningitis, and the like;

Any of such methods can optionally comprise administering an effectiveamount of at least one composition or pharmaceutical compositioncomprising at least one anti-IL-6 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy.Indications for treatment with ant-IL-6 therapy are disclosed in thefollowing references, hereby incorporated by reference into the presentapplication: Van Snick, “Interleukin-6: An Overview,” Ann. Rev.Immunol., 8:253-278 (1990); Campbell et al., “Essential Role forInterferon-gamma And Interleukin-6 in Autoimmune Insulin-DependentDiabetes in NOD/Wehi Mice,” J. Clin. Invest., 87:739-742 (1991);Heinrich et al., “Interleukin-6 Monoclonal Antibody Therapy for aPatient with Plasma Cell Leukemia,” Blood, 78(5):1198-1204 (1991);Starnes et al., “Anti-IL-6 Monoclonal Antibodies Protect Against LethalEscherichia coli Infection and Lethal Tumor Necrosis Factor-alpha.Challenge in Mice,” J. Immunol., 145(12):4185-4191 (1990); Strassman etal., “Evidence for the Involvement of Interleukin 6 in ExperimentalCancer Cachexia,” J. Clin. Invest., 89:1681-1684 (1992).

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-IL-6 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy. Sucha method can optionally further comprise co-administration orcombination therapy for treating such immune diseases or malignantdiseases, wherein the administering of said at least one anti-IL-6antibody, specified portion or variant thereof, further comprisesadministering, before concurrently, and/or after, at least one selectedfrom at least one TNF antagonist (e.g., but not limited to a TNFantibody or fragment, a soluble TNF receptor or fragment, fusionproteins thereof, or a small molecule TNF antagonist), an IL-18 antibodyor fragment, small molecule IL-18 antagonist or IL-18 receptor bindingprotein, an IL-1 antibody (including both IL-1 alpha and IL-1 beta) orfragment, a soluble IL-1 receptor antagonist, an antirheumatic (e.g.,methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, goldsodium thiomalate, hydroxychloroquine sulfate, leflunomide,sulfasalazine, radiation therapy, an anti-angiogenic agent, achemotherapeutic agent, Thalidomide, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial(e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a fluororquinolone, a macrolide, apenicillin, a sulfonamide, a tetracycline, another antimicrobial), anantipsoriatic, a corticosteriod, an anabolic steroid, a diabetes relatedagent, a mineral, a nutritional, a thyroid agent, a vitamin, a calciumrelated hormone, an erythropieitin (e.g., epoetin alpha), a filgrastim(e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2^(nd) Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which references are entirely incorporatedherein by reference. TNF antagonists suitable for compositions,combination therapy, co-administration, devices and/or methods of thepresent invention (further comprising at least one anti body, specifiedportion and variant thereof, of the present invention), include, but arenot limited to, anti-TNF antibodies, antigen-binding fragments thereof,and receptor molecules which bind specifically to TNF; compounds whichprevent and/or inhibit TNF synthesis, TNF release or its action ontarget cells, such as thalidomide, tenidap, phosphodiesterase inhibitors(e.g, pentoxifylline and rolipram), A2b adenosine receptor agonists andA2b adenosine receptor enhancers; compounds which prevent and/or inhibitTNF receptor signalling, such as mitogen activated protein (MAP) kinaseinhibitors; compounds which block and/or inhibit membrane TNF cleavage,such as metalloproteinase inhibitors; compounds which block and/orinhibit TNF activity, such as angiotensin converting enzyme (ACE)inhibitors (e.g., captopril); and compounds which block and/or inhibitTNF production and/or synthesis, such as MAP kinase inhibitors.

Therapeutic Treatments

Any method of the present invention can comprise a method for treatingan IL-6 mediated disorder, comprising administering an effective amountof a composition or pharmaceutical composition comprising at least oneanti-IL-6 antibody to a cell, tissue, organ, animal or patient in needof such modulation, treatment or therapy. Such a method can optionallyfurther comprise co-administration or combination therapy for treatingsuch immune diseases, wherein the administering of said at least oneanti-IL-6 antibody, specified portion or variant thereof, furthercomprises administering, before concurrently, and/or after, at least oneagent as described above.

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one anti-IL-6antibody composition that total, on average, a range from at least about0.01 to 500 milligrams of at least one anti-IL-6 antibody per kilogramof patient per dose, and preferably from at least about 0.1 to 100milligrams antibody/kilogram of patient per single or multipleadministration, depending upon the specific activity of contained in thecomposition. Alternatively, the effective serum concentration cancomprise 0.1-5000 μg/ml serum concentration per single or multipleadministration. Suitable dosages are known to medical practitioners andwill, of course, depend upon the particular disease state, specificactivity of the composition being administered, and the particularpatient undergoing treatment. In some instances, to achieve the desiredtherapeutic amount, it can be necessary to provide for repeatedadministration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5,5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10,10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5,15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19,19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 μg/ml serum concentration per single or multipleadministration, or any range, value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to 10milligrams per kilogram per administration or in sustained release formis effective to obtain desired results. As a non-limiting example,treatment of humans or animals can be provided as a one-time or periodicdosage of at least one antibody of the present invention 0.1 to 100mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one ofday 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, or 40, or alternatively or additionally, at least one of week 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52, or alternativelyor additionally, at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 years, or any combination thereof,using single, infusion or repeated doses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

Parenteral Formulations and Administration

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion or lyophilized powder in association, orseparately provided, with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 1-10% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by known or suitabletechniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent such as aquous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthtetic mono- or di- or tri-glycerides. Parental administrationis known in the art and includes, but is not limited to, conventionalmeans of injections, a gas pressured needle-less injection device asdescribed in U.S. Pat. No. 5,851,198, and a laser perforator device asdescribed in U.S. Pat. No. 5,839,446 entirely incorporated herein byreference.

Alternative Delivery

The invention further relates to the administration of at least oneanti-IL-6 antibody by parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.At least one anti-IL-6 antibody composition can be prepared for use forparenteral (subcutaneous, intramuscular or intravenous) or any otheradministration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms such as, but not limited to, creams and suppositories;for buccal, or sublingual administration such as, but not limited to, inthe form of tablets or capsules; or intranasally such as, but notlimited to, the form of powders, nasal drops or aerosols or certainagents; or transdermally such as not limited to a gel, ointment, lotion,suspension or patch delivery system with chemical enhancers such asdimethyl sulfoxide to either modify the skin structure or to increasethe drug concentration in the transdermal patch (Junginger, et al. In“Drug Permeation Enhancement”; Hsieh, D. S., Eds., pp. 59-90 (MarcelDekker, Inc. New York 1994, entirely incorporated herein by reference),or with oxidizing agents that enable the application of formulationscontaining proteins and peptides onto the skin (WO 98/53847), orapplications of electric fields to create transient transport pathwayssuch as electroporation, or to increase the mobility of charged drugsthrough the skin such as iontophoresis, or application of ultrasoundsuch as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the abovepublications and patents being entirely incorporated herein byreference).

Pulmonary/Nasal Administration

For pulmonary administration, preferably at least one anti-IL-6 antibodycomposition is delivered in a particle size effective for reaching thelower airways of the lung or sinuses. According to the invention, atleast one anti-IL-6 antibody can be delivered by any of a variety ofinhalation or nasal devices known in the art for administration of atherapeutic agent by inhalation. These devices capable of depositingaerosolized formulations in the sinus cavity or alveoli of a patientinclude metered dose inhalers, nebulizers, dry powder generators,sprayers, and the like. Other devices suitable for directing thepulmonary or nasal administration of antibodies are also known in theart. All such devices can use of formulations suitable for theadministration for the dispensing of antibody in an aerosol. Suchaerosols can be comprised of either solutions (both aqueous and nonaqueous) or solid particles. Metered dose inhalers like the Ventolin®metered dose inhaler, typically use a propellent gas and requireactuation during inspiration (See, e.g., WO 94/16970, WO 98/35888). Drypowder inhalers like Turbuhaler™ (Astra), Rotahaler® (Glaxo), Diskus®(Glaxo), Spiros™ inhaler (Dura), devices marketed by InhaleTherapeutics, and the Spinhaler® powder inhaler (Fisons), usebreath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP237507 Astra, WO 97/25086 Glaxo, WO 94/08552. Dura, U.S. Pat. No.5,458,135 Inhale, WO 94/06498 Fisons, entirely incorporated herein byreference). Nebulizers like AERx™ Aradigm, the Ultravent® nebulizer(Mallinckrodt), and the Acorn II® nebulizer (Marquest Medical Products)(U.S. Pat. No. 5,404,871 Aradigm, WO 97/22376), the above referencesentirely incorporated herein by reference, produce aerosols fromsolutions, while metered dose inhalers, dry powder inhalers, etc.generate small particle aerosols. These specific examples ofcommercially available inhalation devices are intended to be arepresentative of specific devices suitable for the practice of thisinvention, and are not intended as limiting the scope of the invention.Preferably, a composition comprising at least one anti-IL-6 antibody isdelivered by a dry powder inhaler or a sprayer. There are a severaldesirable features of an inhalation device for administering at leastone antibody of the present invention. For example, delivery by theinhalation device is advantageously reliable, reproducible, andaccurate. The inhalation device can optionally deliver small dryparticles, e.g. less than about 10 μm, preferably about 1-5 μm, for goodrespirability.

Administration of IL-6 Antibody Compositions as a Spray

A spray including IL-6 antibody composition protein can be produced byforcing a suspension or solution of at least one anti-IL-6 antibodythrough a nozzle under pressure. The nozzle size and configuration, theapplied pressure, and the liquid feed rate can be chosen to achieve thedesired output and particle size. An electrospray can be produced, forexample, by an electric field in connection with a capillary or nozzlefeed. Advantageously, particles of at least one anti-IL-6 antibodycomposition protein delivered by a sprayer have a particle size lessthan about 10 μm, preferably in the range of about 1 μm to about 5 μm,and most preferably about 2 μm to about 3 μm.

Formulations of at least one anti-IL-6 antibody composition proteinsuitable for use with a sprayer typically include antibody compositionprotein in an aqueous solution at a concentration of about 0.1 mg toabout 100 mg of at least one anti-IL-6 antibody composition protein perml of solution or mg/gm, or any range or value therein, e.g., but notlimited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml ormg/gm. The formulation can include agents such as an excipient, abuffer, an isotonicity agent, a preservative, a surfactant, and,preferably, zinc. The formulation can also include an excipient or agentfor stabilization of the antibody composition protein, such as a buffer,a reducing agent, a bulk protein, or a carbohydrate. Bulk proteinsuseful in formulating antibody composition proteins include albumin,protamine, or the like. Typical carbohydrates useful in formulatingantibody composition proteins include sucrose, mannitol, lactose,trehalose, glucose, or the like. The antibody composition proteinformulation can also include a surfactant, which can reduce or preventsurface-induced aggregation of the antibody composition protein causedby atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbitol fatty acidesters. Amounts will generally range between 0.001 and 14% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan monooleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein such as IL-6 antibodies, or specified portionsor variants, can also be included in the formulation.

Administration of IL-6 Antibody Compositions by a Nebulizer

Antibody composition protein can be administered by a nebulizer, such asjet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer,a compressed air source is used to create a high-velocity air jetthrough an orifice. As the gas expands beyond the nozzle, a low-pressureregion is created, which draws a solution of antibody compositionprotein through a capillary tube connected to a liquid reservoir. Theliquid stream from the capillary tube is sheared into unstable filamentsand droplets as it exits the tube, creating the aerosol. A range ofconfigurations, flow rates, and baffle types can be employed to achievethe desired performance characteristics from a given jet nebulizer. Inan ultrasonic nebulizer, high-frequency electrical energy is used tocreate vibrational, mechanical energy, typically employing apiezoelectric transducer. This energy is transmitted to the formulationof antibody composition protein either directly or through a couplingfluid, creating an aerosol including the antibody composition protein.Advantageously, particles of antibody composition protein delivered by anebulizer have a particle size less than about 10 μm, preferably in therange of about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm.

Formulations of at least one anti-IL-6 antibody suitable for use with anebulizer, either jet or ultrasonic, typically include a concentrationof about 0.1 mg to about 100 mg of at least one anti-IL-6 antibodyprotein per ml of solution. The formulation can include agents such asan excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the at least one anti-IL-6antibody composition protein, such as a buffer, a reducing agent, a bulkprotein, or a carbohydrate. Bulk proteins useful in formulating at leastone anti-IL-6 antibody composition proteins include albumin, protamine,or the like. Typical carbohydrates useful in formulating at least oneanti-IL-6 antibody include sucrose, mannitol, lactose, trehalose,glucose, or the like. The at least one anti-IL-6 antibody formulationcan also include a surfactant, which can reduce or preventsurface-induced aggregation of the at least one anti-IL-6 antibodycaused by atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbital fatty acidesters. Amounts will generally range between 0.001 and 4% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan mono-oleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein such as antibody protein can also be includedin the formulation.

Administration of IL-6 Antibody Compositions by a Metered Dose Inhaler

In a metered dose inhaler (MDI), a propellant, at least one anti-IL-6antibody, and any excipients or other additives are contained in acanister as a mixture including a liquefied compressed gas. Actuation ofthe metering valve releases the mixture as an aerosol, preferablycontaining particles in the size range of less than about 10 μm,preferably about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm The desired aerosol particle size can be obtained byemploying a formulation of antibody composition protein produced byvarious methods known to those of skill in the art, includingjet-milling, spray drying, critical point condensation, or the like.Preferred metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant.

Formulations of at least one anti-IL-6 antibody for use with ametered-dose inhaler device will generally include a finely dividedpowder containing at least one anti-IL-6 antibody as a suspension in anon-aqueous medium, for example, suspended in a propellant with the aidof a surfactant. The propellant can be any conventional materialemployed for this purpose, such as chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydro fluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the like.Preferably the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one anti-IL-6 antibody as a suspensionin the propellant, to protect the active agent against chemicaldegradation, and the like. Suitable surfactants include sorbitantrioleate, soya lecithin, oleic acid, or the like. In some casessolution aerosols are preferred using solvents such as ethanol.Additional agents known in the art for formulation of a protein such asprotein can also be included in the formulation.

One of ordinary skill in the art will recognize that the methods of thecurrent invention can be achieved by pulmonary administration of atleast one anti-IL-6 antibody compositions via devices not describedherein.

Oral Formulations and Administration

Formulations for oral administration rely on the co-administration ofadjuvants (e.g., resorcinols and nonionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) toincrease artificially the permeability of the intestinal walls, as wellas the co-administration of enzymatic inhibitors (e.g., pancreatictrypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) toinhibit enzymatic degradation. The active constituent compound of thesolid-type dosage form for oral administration can be mixed with atleast one additive, including sucrose, lactose, cellulose, mannitol,trehalose, raffinose, maltitol, dextran, starches, agar, arginates,chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin,collagen, casein, albumin, synthetic or semisynthetic polymer, andglyceride. These dosage forms can also contain other type(s) ofadditives, e.g., inactive diluting agent, lubricant such as magnesiumstearate, paraben, preserving agent such as sorbic acid, ascorbic acid,.alpha.-tocopherol, antioxidant such as cysteine, disintegrator, binder,thickener, buffering agent, sweetening agent, flavoring agent, perfumingagent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No.5,871,753 are used to deliver biologically active agents orally areknown in the art.

Mucosal Formulations and Administration

For absorption through mucosal surfaces, compositions and methods ofadministering at least one anti-IL-6 antibody include an emulsioncomprising a plurality of submicron particles, a mucoadhesivemacromolecule, a bioactive peptide, and an aqueous continuous phase,which promotes absorption through mucosal surfaces by achievingmucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucoussurfaces suitable for application of the emulsions of the presentinvention can include corneal, conjunctival, buccal, sublingual, nasal,vaginal, pulmonary, stomachic, intestinal, and rectal routes ofadministration. Formulations for vaginal or rectal administration, e.g.suppositories, can contain as excipients, for example,polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulationsfor intranasal administration can be solid and contain as excipients,for example, lactose or can be aqueous or oily solutions of nasal drops.For buccal administration excipients include sugars, calcium stearate,magnesium stearate, pregelinatined starch, and the like (U.S. Pat. No.5,849,695).

Transdermal Formulations and Administration

For transdermal administration, the at least one anti-IL-6 antibody isencapsulated in a delivery device such as a liposome or polymericnanoparticles, microparticle, microcapsule, or microspheres (referred tocollectively as microparticles unless otherwise stated). A number ofsuitable devices are known, including microparticles made of syntheticpolymers such as polyhydroxy acids such as polylactic acid, polyglycolicacid and copolymers thereof, polyorthoesters, polyanhydrides, andpolyphosphazenes, and natural polymers such as collagen, polyaminoacids, albumin and other proteins, alginate and other polysaccharides,and combinations thereof (U.S. Pat. No. 5,814,599).

Prolonged Administration and Formulations

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot or implant dosage forms can be utilized. Forexample, a dosage form can contain a pharmaceutically acceptablenon-toxic salt of the compounds that has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) asalt with a polyvalent metal cation such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and thelike, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g. gas orliquid liposomes are known in the literature (U.S. Pat. No. 5,770,222and “Sustained and Controlled Release Drug Delivery Systems”, J. R.Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

ABBREVIATIONS

BSA—bovine serum albuminEIA—enzyme immunoassayFBS—fetal bovine serumH₂O₂-hydrogen peroxideHRP—horseradish peroxidase\Ig—immunoglobulin

IL-6-Interleukin-6

IP—intraperitonealIV—intravenousMab—monoclonal antibodyOD—optical densityOPD—o-Phenylenediamine dihydrochloridePEG—polyethylene glycolPSA—penicillin, streptomycin, amphotericinRT—room temperatureSQ—subcutaneousv/v—volume per volumew/v—weight per volume

Example 1 Generation of Murine CLB8 Mab Immunization

The hybridoma giving rise to the murine CLB-IL6-8 antibody was derivedfrom a fusion performed in a laboratory of Dr. Lucien Aarden, CentralLaboratory of the Netherlands Red Cross Transfusion Service (CLB) asreported (Brackenhoff et al, J. Immunol. (1990) 145: 561-568).

Eight week old female Balb/c mice obtained from CLB's specified pathogenfree breeding stock were immunized intramuscularly (IM) with 10 μg ofpurified recombinant interleukin-6 (rIL-6) (CLB) emulsified in CompleteFreund's adjuvant. Three subsequent IM injections with 10 μg each ofrIL-6 in Incomplete Freund's adjuvant were carried out at intervals of4-8 weeks.

Cell Fusion

Four days after the last IM booster injection, a mouse was sacrificed;the spleen was removed and finely minced. A single cell suspension wasobtained in ambient Earle's balanced salt solution. The cells werewashed and counted. A fusion was carried out at a 1:3 ratio of viablespleen cells to murine myeloma cells (SP2/0-Ag14) in the presence of 42%(w/v) polyethylene glycol in Iscove's modified Dulbecco's medium (IMDM).The non-Ig secreting fusion partner SP2/0 was established from a cellbank maintained at CLB. After fusion, cells were resuspended in IMDM,supplemented with 5% fetal bovine serum, 50 μM penicillin/streptomycin,5×10⁻⁵ M 2-mrecaptoethanol (2-ME) and HAT (6×10⁻⁴ M hypoxanthine,6.5×10⁻⁷ M aminopterin, 6.4×10⁻⁵ M thymidine). The proliferation ofthese hybridomas immediately after fusion is dependent on IL-6,therefore, 100 U/mL of purified murine IL-6 (Van Smick, Brussels) wasadded to the selection medium. The fused cells were then distributedinto 96-well plates at 1×10⁵ cells/100 microL well.

Primary Characterization of Murine Anti IL-6 Hybridomas

Anti-IL-6 secreting hybrids were selected by enzyme linked immunosorbentassay (ELISA) and radioimmunoassay (RIA) (Brackenhoff et al. (1990) 145:561-568).

A solid phase ELISA was employed for screening monoclonal antibodiesspecific for human IL-6. Purified rIL-6 (0.5 μg/mL) was coated overnightat room temperature in phosphate buffered saline (PBS) on flat-bottomedplates (Dynatech), 100 μL/well. The plates were washed with PBS, 0.02%(v/v) Tween 20 (PBS/Tween) and were incubated with 1:2 dilutions ofculture supernatants in PBS/Tween supplemented with 0.2% gelatin (PTG)for 2 hours at ambient temperature. After washing, the plates wereincubated with horseradish peroxidase-conjugated monoclonal ratanti-mouse kappa light chain 226 (Einstein University, NY) in PTG (2μg/mL) for 1 hour. The plates were washed and the bound peroxidase wasdetected with 100 μL/well of 3,5,3,5,tetramethylbenzidine/0.003%hydrogen peroxide in 0.1-M sodium acetate, pH 5.5. The color reactionwas stopped with 2M H₂SO₄ and the plates were read at 450 nM on aTitertek, Multiscan reader. The wells yielding positive OD's werechosen.

A solid phase RIA was also employed for screening anti-IL-6 hybridomas.Goat anti-murine Ig antibodies were coupled to cyanogen-bromideactivated sepharose CL-4B (Pharmacia). The sepharose was washed andresuspended at 10 mg/mL in PBS, 0.1% Tween 20, 0.1% sodium azide.Hybridoma supernatants were added to sepharose beads in the presence ofapproximately 20,000 counts/minute of ¹²⁵Iodine-rIL-6 (CLB) for 6 hourswith constant mixing. The beads were washed extensively in PBS/Tween andcounted in a gamma counter. The wells yielding the highest specificactivities were chosen.

Hybridomas that were positive in both assay systems were established andsubcloned twice at limiting dilutions in IMDM supplemented with 2×10⁻⁵ M2-ME and 5% FBS (complete IMDM). The IL-6 independent subclone CLB-IL6-8was selected and maintained in complete IMDM. Stock cultures testednegative for mycoplasma using an indirect Hoescht stain after 4 days inculture on Vero76 target cells. Isotype determination of supernatant viaInnogenetics Line ImmunoAssay (INNO-LIA) mouse monoclonal antibodyisotype kit yielded a single murine isotype IgG₁ kappa. This isotypedetermination was confirmed by a capture EIA

The murine hybridoma and cell line was so produced, CLBIL-6/8 was calledCLB8. It was chimerized and further characterized as described below.

Example 2 Chimerization and Sequencing

Cloning and Expression of the cCLB8 Variable Region Genes

Genomic DNA was isolated from the murine hybridoma C143A which secretesa murine monoclonal antibody specific for human IL-6.

For the light chain, the DNA was digested with restriction endonucleaseHind III and subjected to electrophoresis through a 0.8% agarose gel.The portion of the gel containing DNA fragments approximately 3.4 Kb inlength was excised, and the DNA was eluted. The fragments were ligatedinto the vector8charon27, and packaged into bacteriophage particles. Forthe heavy chain, the DNA was digested with restriction endonuclease EcoR¹ and subjected to electrophoresis through a 0.8% agarose gel. Theportion of the gel containing DNA fragments approximately 3.6 Kb inlength was excised, and the DNA was eluted. The fragments were ligatedinto the vector8gt10, and packaged into bacteriophage particles.

Both heavy and light chain bacteriophage libraries were plated on E.Coli, and grown overnight. The plaques were transferred tonitrocellulose filters, and probed with ³²P-labeled DNA fragmentscorresponding to murine J_(κ) (light chain) or murine J_(H) sequences.Positive plaques were identified and plaque purified. Phage DNA wasisolated, and the Hind III (light chain) or Eco RI (heavy chain) insertswere isolated and cloned into immunoglobulin expression vectors.

Heavy and light chain expression plasmids were used to cotransfect SP2/0cells, and mycophenolic acid selection was applied. Individual clonesproducing chimeric antibody were identified and subcloned to insuremonoclonality and to generate higher producers.

Antibody purified from individual cell lines was tested forneutralization ability in an IL-6-dependent B9 cell proliferation assay.The antibody is referred as chimeric CLB8 or cCLB8 throughout thisapplication.

cCLB8 Heavy Chain Variable RegionE   V   Q   L   V   E   S   G   G   K   L   L   K   P   G   G   S   L   K   LGAG GTG CAA CTG GTG GAA TCT GGA GGA AAA TTA CTG AAG CCT GGA GGG TCC CTG AAA CTCS   C   A   A   S   G   F   T   F   S   S   F   A   M   S   W   F   R   Q   STCC TGT GCA GCC TCT GGA TTC ACC TTC AGT AGC TTT GCC ATG TCT TGG TTT CGC CAG TCT                                                                        CDR 1 P   E   K   R   L   E   W   V   A   E   I   S   S   G   G   S   Y   T   Y   YCCA GAG AAG AGG CTG GAG TGG GTC GCA GAA ATT AGT AGT GGT GGG AGT TAC ACC TAC TAT      CDR 2 P   D   T   V   T   G   R   F   T   I   S   R   D   N   A   K   N   T   L   YCCT GAC ACT GTG ACG GGC CGA TTC ACC ATC TCC AGA GAC AAT GCC AAG AAC ACC CTG TACL   E   M   S   S   L   R   S   E   D   T   A   M   Y   Y   C   A   R   G   LCTG GAA ATG AGC AGT CTG AGG TCT GAG GAC ACG GCC ATG TAT TAT TGT GCA AGG GGT TTA W   G   Y   Y   A   L   D   Y   W   G   Q   G   T   S   V   T   V   S   S  TGG GGG TAC TAT GCT CTT GAC TAC TGG GGT CAA GGA ACC TCA GTC ACC GTC TCC TCA       CDR 3 cCLB8 Light Chain Variable Region Q   I   V   L   I   Q   S   P   A   I   M   S   A   S   P   G   E   K   V   TCAA ATT GTT CTC ATA CAG TCT CCA GCA ATC ATG TCT GCA TCT CCA GGG GAG AAG GTC ACC M   T   C   S   A   S   S   S   V   S   Y   M   Y   W   Y   Q   Q   K   P   GATG ACC TGC AGT GCC AGC TCA AGT GTA AGT TAC ATG TAC TGG TAC CAG CAG AAG CCA GGACDR 1 S   S   P  R   L   L   I   Y  D   T   S   N   L   A  S   G   V   P   V  RTCC TCC CCC AGA CTC CTG ATT TAT GAC ACA TCC AAC CTG GCT TCT GGA GTC CCT GTT CGC                                                         CDR 2F   S   G   S   G   S   G   T   S   Y   S   L   T   I   S   R   M   E   A   ETTC AGT GGC AGT GGG TCT GGG ACC TCT TAC TCT CTC ACA ATC AGC CGA ATG GAG GCT GAGD   A   A   T   Y   Y   C   Q   Q   W   S   G   Y   P   Y   T   F   G   G   GGAT GCT GCC ACT TAT TAC TGC CAG CAG TGG AGT GGT TAC CCA TAC ACG TTC GGA GGG GGG                                                CDR 3 T   K   L   E   I   K ACC AAG CTG GAA ATA AAA

Example 3 Measure of Binding of cCLB8 to Human IL-6 by Solid Phase EIA

A solid phase EIA was used to assess binding characteristics of cCLB8Mab to human IL-6. Briefly, plates were coated with recombinant humanIL-6 (RDI) at 1 μg/mL in PBS overnight at 4° C. After washing in 0.15Msaline containing 0.02% (v/v) Tween 20, the wells were blocked with 1%(w/v) BSA in PBS, 200 μL/well for 1 hour at RT. Purified antibody wasincubated in two-fold serial dilutions from a starting concentration of5 μg/mL for 1 hour at 37° C. The plate was washed and then probed with50 μL/well HRP-labeled goat anti-human IgG (Tago) diluted 1:20,000 in 1%BSA-PBS for 1 hour at RT. The plate was again washed and 100 μL/well ofthe citrate-phosphate substrate solution (0.1M citric acid and 0.2Msodium phosphate, 0.01% H₂O₂ and 1 mg/mL OPD) was added for 15 minutesat RT. Stop solution (4N sulfuric acid) was then added at 25 μL/well andthe absorption at 490 nm quantitated using an automated platephotometer. FIG. 1 shows cCLB8 binding to IL-6 measured as OD 490 nmdemonstrating that cCLB8 binds to recombinant human IL-6 in aconcentration dependent manner.

Example 4 In Vitro Neutralization Assays

Blockade of IL-6 by cCLB8 Inhibits Secretion of IgM and MCP-1

The chimeric monoclonal antibody cCLB8 was assessed in two simplebioassay formats to determine its neutralizing bioactivity on IL6induced secretion of human IgM and the chemokine MCP-1. Two human celllines were used in these studies. The SKW6.4 cell line was originallyderived from an EBV transformed Burkitt's B cell lymphoma and secretessoluble IgM in response to IL6. The U937 cell line is monoblastic,committed to monocyte differentiation and was originally isolated from apatient with diffuse histiocytic lymphoma. U937 cells secrete MCP-1 inresponse to IL6. cCLB8 inhibition of these particular bioactivities wereevaluated since they could easily be monitored using an EIA format.Prior to assay, cells were serum starved over night and then culturedthe following day alone, with IL6 or with IL6 preincubated with variousconcentrations of antibody or a negative control antibody. At theconclusion of a 72 hour incubation supernatants were collected and usedin IgM specific and MCP-1 specific ETA's. The results as shown in FIGS.2 and 3, demonstrate cCLB8 significantly inhibits IL6 mediated secretionof IgM and MCP-1 in vitro.

In the experiment represented by FIG. 2, EIA plates were coated withgoat anti-human IgM Fc5μ fragment specific in 10 mM carbonate buffer, pH9.6 overnight at 4° C. Plates were washed with 0.15 M saline with 0.02%v/v Tween 20 and blocked with PBS/1% w/v BSA for 1 hour. Cell culturesupernatant was added at serial two-fold dilutions. Following incubationand subsequent washes with 0.02% Tween, 0.15M saline the plate wasprobed with HRP-labeled goat anti-human IgMμ chain specific. OPDsubstrate was then added and following color development, OD read at 490nm. The data indicate that cCLB8, but not the isotype matched negativecontrol chimeric mAb, inhibits IgM mu secretion.

In the experiment represented by FIG. 3, EIA plates were coated withgoat anti-human MCP-1 in 10 mM carbonate buffer, pH 9.6 overnight at 4°C. Plates were washed with 0.15 M saline with 0.02% v/v Tween 20 andblocked with PBS/1% w/v BSA for 1 hour. Cell culture supernatant wasadded at serial two-fold dilutions. Following a two-hour incubation andsubsequent washes with 0.02% Tween, 0.15M saline, the plate was probedwith biotinylated anti-human MCP-1 as per manufacturer's instructions.Plates were washed again and HRP-labeled streptavidin was added for 1hour. Color development was done with TMB substrate. OD read at 450. Thedata indicate that cCLB8, but not cK931, inhibit IL-6 mediated MCP-1Production.

Neutralization of IL6-Mediated Phosphorylation of STAT3.

The IL6 receptor consists of an 80 kD binding sub-unit, IL6Rα and thesignal transduction sub-unit, gp130. IL6 binds to the IL6Rα sub-unit andinitiates the association of IL6Rα and gp130 resulting in a highaffinity receptor and signal transduction. IL6Rα also exists in asoluble form. IL6 can bind to soluble IL6R (sIL6R) and the complex canact on cells expressing gp130. IL6 has been shown to activate STAT3. Ahuman acute monocytic leukemia cell line, THP-1, was used to demonstrateinhibition of STAT3 phosphorylation by cCLB8. Cells were stimulated with(IL6+sIL6R)+/−cCLB8 or irrelevant antibody (K931) as a negative control.Cell lysates were immunoprecipitated with anti-STAT3, samples resolvedon 7.5% SDS-PAGE and transferred to Hybond-P membrane, followed byWestern blotting using anti-phosphotyrosine-HRP. ECLplus was used fordetection.

The data represented in FIG. 4 show that cCLB8 can inhibitphosphorylation of STAT3 when it is a) allowed to bind to rhIL6 beforeaddition of sIL6R, b) allowed to bind to sIL6R before addition of rhIL6,or c) when rhIL6 is allowed to bind to sIL6R before addition of cCLB8and cells. THP-1 cells incubated in media without serum for 16 hr,scraped from flasks and resuspended in 0.5 ml media Lanes 2-6, IL6incubated+/−antibody for 15 min, then sIL6R added and incubated for 15min. Cells added and incubated additional 15 min. Lane 7, IL6 incubatedwith sIL6R for 15 min, then CLB-IL6 added and incubated for 15 min.Cells added and incubated additional 15 min. Samples immunoprecipitatedwith anti-STAT3 [1 μg/m1], resuspended in Laemmli sample buffer andresolved on 7.5% SDS-PAGE and transferred to Hybond-P. Membraneincubated with anti-phosphotyrosine-HRP.

cCLB8 Inhibits Serum Amyloid A Production

IL-1β is a potent inducer of Serum Amyloid A (SAA) production from HepG2human hepatoma cells in the presence of human IL-6 (Smith and McDonald,Clin Exp. Immunol. 90:293-9 (1992)). Therefore, cCLB8 Mab was assayedfor the ability to inhibit IL-1β/IL-6 induced SAA production from thesecells. Briefly, HepG2 cells were seeded at 2.25×10⁵/well for 24 hours.IL-6 (100 ng/ml, RDI) and IL-6sR (200 ng/ml, S & D) were preincubatedfor 30 minutes, and mixed with IL-1β (1 ng/ml, R&D). The cCLB8 Mab and anegative isotype control Mab (cSF25) were serially diluted and thenpreincubated with the above mixture for 30 minutes more.

For the experimental results shown in FIG. 5, serial dilutions of cCLB8or cSF25 (an isotype matched irrelevant Mab) were preincubated withIL-6sR and IL-1β and then cultured with HepG2 cells for 24 hours. Cellsupernatant was then analyzed for Serum amyloid A production levels byELISA. (Human SAA ELISA kit, Biosource, performed according tomanufacturer's instructions). Error bars indicate SEM of duplicatesamples. The data in FIG. 5 indicate that cCLB8 was able to inhibitIL-1β/IL-6 induced SAA production from HepG2 cells in a dose dependentmanner.

Inhibition of IL-6 Induced Cell Proliferation by cCLB8 Mab

The murine B myeloma cell line, 7TD1, is induced to proliferate in thepresence of IL-6. To demonstrate the ability of cCLB8 Mab to neutralizethe activity of IL-6, the cells were incubated at 37° C. for 72 hours inIMDM containing 10% FBS and 0.5 ng/mL recombinant human IL-6 (R&DSystems), and with serial dilutions of cCLB8 Mab or negative control Mab17-1A. Cell proliferation was measured by a luminescent ATP assay(ATPLite, Packard Bioscience) which correlates directly with cellnumber.

The data shown in FIG. 6 demonstrate that IL-6 dramatically stimulatesproliferation of 7TD1 cells and cCLB8 inhibited this cell proliferationin a concentration dependent manner with an EC₅₀ of 7.2 ng/mL. Errorbars indicate the SEM of duplicate samples. * represents proliferationof cells in the absence of rIL-6.

Example 5 Epitope Mapping

The epitopes of several neutralizing anti IL-6 Mabs including CLB8 havebeen characterized using antibody binding to human IL-6 mutant proteinsas described in (Brakenhoff, J. et al. (1990) J. Immunology 145:561-568). Amino and carboxyl-terminal deletion mutants were prepared andthe panel of antibodies to IL-6 was analyzed by antibody competitionexperiments. On the basis of the competition studies the neutralizingMabs were divided into 2 groups (I and II). In this method, residuesincluded in the epitope of a given Mab are delineated by its failure torecognize the corresponding site-specific single amino acid substitutionvariants of the antigenic protein. CLB.IL-6/8 was mapped to site I onthe human IL-6 molecule which is composed of amino acids Gln29-Leu34 inclose proximity of the carboxyl terminus of the molecule. Furtherstudies (Kalai, M, et al., Eur. J. Biochem. 249, 690-700 (1997)) showedthat CLB. IL-6/8 recognized amino acid residues crucial for the bindingof IL-6 to the IL-6R (gp80). These studies also indicated that itsepitope covers the ends of both the AB loop and the D helix regions ofthe IL-6 molecule.

Example 6 In Vivo Characterization

Treatment with Anti-Human IL-6 (cCLB8) and Anti-Mouse IL-6 Mouse MabsDelays Cancer Cachexia

Human melanoma cells (A375S2) were inoculated into female nude mice andMab therapy was initiated on the same day. Antibodies were injectedintraperitoneally at a dose of 10 mg/kg (2×/week) and C57 (anti-CMV) wasused as a control mAb. Combination of cCLB8 (anti-human IL-6) andMP520F3 (Mab to mouse IL-6, R&D Systems) were used to create a combinedblockade significantly inhibited weight loss of human melanoma tumorbearing animals compared to control antibody C57 treated animals (FIG.7). Antibody therapy did not effect tumor growth or final tumor weight.These findings indicate that IL6 participates in tumor-induced animalweight loss, and blockade of IL-6 can delay cancer cachexia in thismodel.

FIG. 7. Combined blockade of human and mouse IL6 (anti-IL6 mAbs cCLB8and MP520F3) results in significant inhibition of animal weight loss.Corrected animal weight loss on Y axis is (animal weight-tumor weight atthe end of the study) minus animal weight at start of the study. Eachbar is the mean of data from at least 14 animals/group and error barsindicate standard deviation. A two tailed t-test analysis indicated thatthe anti-IL-6 group significantly inhibited body weight loss withp=0.007.

Example 7 Affinity Measurements

BIAcore 2000, Sensor Chip CM-5 (gold surface on chip covered with acarboxymethylated dextran matrix), HBS (10 mM HEPES with 0.15 M NaCl,3.4 mM EDTA, and 0.05% surfactant P20 at pH 7.4), amine couplingreagents (N-hydroxysuccinimde (NHS),N-ethyl-N′-(3-methylaminopropyl)-carbodimiide (EDC) and 1M ethanolamineHCl) were obtained from BIAcore and prepared according to themanufacturer's instructions. Anti-human Fc (Jackson AffiniPure Goatanti-human IgG, Fc, Cat#109-005-098, Lot#48646) was purchased fromJackson ImmunoResearch.

Chimeric CLB8 (Lot# PD1F03) IgG monoclonal antibody in 5 ml of 0.15MSodium Chloride, 0.01M Sodium Phosphate, pH 7.2 was manufactured byCentocor. Recombinant Human IL-6 (Lot# A1197111) was purchased from R&DSystems.

An anti-human Fc (1.8 mg/ml) was diluted to a concentration of 50 μg/mlin NaOAc buffer (10 mM, pH 4.8) and coupled to the carboxymethylateddextran matrix of a CM-5 sensor chip using the manufacturer'samine-coupling chemistry as described in the BIAcore systems manual.Using the surface preparation wizard aiming for 10000 RU, the carboxylgroups on the sensor surfaces were first activated with NHS/EDC followedby the addition of the anti-human Fc. The remaining activated groupswere blocked by the injection of 1M ethanolamine. Each of the flow cellswas coupled individually. Employing these conditions, the four flow cellsurfaces containing 7554-9571 resonance units (RU) of anti-human Fc wereprepared. In preliminary experiments, it was determined that threeinjections (15 ul at 30 μl/min) 100 mM H3PO4/0.05% CHAPS wouldefficiently remove the bound immunoglobulin and preserve the bindingcapacity of the immobilized anti-human Fc.

Two experiments were performed on the BIAcore 2000 at 25° C. and a flowrate of 30 μL/min. cCLB8 was dissolved in HBS at 5 ug/ml. The analyte,IL-6, was dissolved in HBS at 0.25, 0.125, 0.062, 0.031, and 0.015μg/ml. A designated amount of antibody was flowed over its respectiveflow cell followed by injections of 30 μl of each IL-6 concentration at30 μl/min (association phase) and an uninterrupted 800 seconds of bufferflow (dissociation phase). The surface of the chip was regenerated bythree sequential injections of 15 μl each with 100 mM H3PO₄/0.05% CHAPS.The injections of HBS serve as a reference (blank sensogram) for thesubtraction of bulk refractive indices for analysis. Using the 1:1 modelin BlAanalysis 3.0, a local fit was done for both dissociation (kd,[s-1] and association (ka, [M-1s-1]) and the dissociation constant (KD,[M]) calculated (kd/ka).

Analysis was done using BIAevaluation version 3.0. Kinetic constantswere derived from sensogram data by fitting the experimental curves tothe rate equations derived from models of the interaction mechanism. Aglobal analysis using a 1:1 binding model with local RUmax fit, the ka,kd, KD were determined (Table 1).

TABLE 1 Affinity Measurements for cCLB8 Mab by Biacore Sample k_(a)(m⁻¹s⁻¹)(×10⁶) k_(d) (s⁻¹) (×10⁻⁵) K_(D)(M) (×10⁻¹¹) Chi² cCLB8 1.1 6.25.7 0.111 cCLB8 0.37 5.2 14 0.236

Example 8 Anti-Idiotype Antibodies

Development of effective assay systems (immunohistochemistry and serumdetection) for cCLB8 requires the use of anti-idiotypic antibodies.Therefore Balb/c mice are immunized with cCLB8 to generateanti-idiotypic antibodies to cCLB8 that may be utilized aspharmacokinetic probes in serum detection and immunohistochemicalassays.

Immunization

Five Balb/c mice (Charles River Laboratories) at 6-7 weeks of age wereimmunized over a 12-week period with cCLB8 (Centocor, PD1F03) given at50 μg IP and 25 μg SC. Each mouse received IP and SC injections.Injections occurred at 2-week intervals throughout the immunizationregimen. Injection material administered IP was emulsified with an equalvolume of Freund's Adjuvant (Sigma). The first IP injection utilizedCompleted Freund's Adjuvant in a total volume of 200 μA Subsequent IPinjections contained Incomplete Freund's Adjuvant. Injection materialadministered SC was diluted in PBS and divided between two injectionsites at 100 μl/site. The mice were bled on days 0, 21, 47, and 77.Blood collections were performed on anesthetized mice by retro-orbitalpuncture, and serum was collected for titer determination by cCLB8 solidphase EIA. Three weeks following the end of the immunization protocol,Mouse #1 received a final IV booster injection of 100 μg cCLB8 dilutedin 125 μl PBS.

Generation of Mouse cCLB8 Anti-Idiotypic Monoclonal Antibodies

One fusion utilizing a cCLB8 immunized Balb/c mouse spleen resulted inidentification of 7 anti-id antibodies specific for cCLB8 via EIA. The 7anti-id antibodies were shown not to bind other mouse/human chimericantibodies such as C207A, C128A, C168J, C116J, C300A, and C301A. Six ofthe seven antibodies were of isotype IgG1κ and one antibody was IgG2bκ.Table 1 summarizes the results of the fusion. It should be noted that amaximum serum titer of 1:800 was achieved in the mouse after 47 days andremained constant throughout the duration of immunization.

Isotyping

Isotype determination of the antibodies was accomplished by use of MouseMonoclonal Antibody Isotyping Kit (Life Technologies) in dipstickformat. A mixture of dilution buffer, hybridoma supernatant, and Ratanti-mouse conjugate were incubated overnight at RT with shaking intubes containing sticks pre-coated with various capture murine antibodyisotypes. Sticks were removed from tubes, rinsed gently in dH₂O, andisotypes determined

TABLE 1 Properties of Mouse cCLB8 anti-idiotypic Monoclonal Antibodies Ccode Isotype C433A IgG1κ C434A IgG1κ C435A IgG1κ C436A IgG2bκ C437AIgG1κ C438A IgG1κ C439A IgG1κ

Serum Inhibition Assays

The effect of pooled normal human serum (NHS) on the 7 anti-idantibodies' ability to bind cCLB8 was determined Doubling dilutions ofAnti-id Mabs starting at 50 μg/ml were incubated in the presence of 0%,0.5%, 5%, and 50% NHS for 30 minutes at 37° C. The mixtures weretransferred to cCLB8 coated plates and incubated for 30 minutes at 37°C. Plates were washed then probed with goat anti-mouse IgG Fc*HRP. Noneof the anti-id Mabs were prevented from binding cCLB8 by 0% and 0.5%NHS. Three Mabs (C433A, C435A, and C437A) exhibited partial bindinginhibition by 5% NHS. All except C434A and C436A were significantlyaffected by NHS concentration of 50% (FIGS. 8 A-G).

Inhibition of cCLB8 Binding to HuIL-6 by Anti-id Mabs

The capability of the 7 anti-id antibodies to inhibit cCLB8 binding toHuIL-6 was assessed. Previous EIA studies have shown that cCLB8 bindsvery weakly to HuIL-6 coated plates. Two Mabs (C435A and C437A) atconcentration excesses of 6-25 fold demonstrated virtually completeinhibition of cCLB8 binding to HuIL-6. C434A expressed an inhibitoryeffect for cCLB8 only at 25-fold excess. The two best antibodies atinhibiting binding of cCLB8 to HuIL-6 were C436A and C439A. These twoantibodies were able to completely inhibit cCLB8 binding over an excessconcentration range of 3 to 25 fold. C433A and C438A showed noinhibitory activity (FIG. 9). This assay confirmed the results obtainedin a preliminary study.

Anti-id Binding to cCLB8 Pre-Bound to HuIL-6

The capacity of the 7 anti-id antibodies to bind cCLB8 that waspre-bound to HuIL-6 was examined. cCLB8 at 10 μg/ml incubated on HuIL-6plates for 30 minutes at 37° C. Plates were washed then incubated withtripling dilutions of Anti-id Mabs starting at 10 μg/ml for 30 minutesat 37° C. Plates were washed then probed with goat anti-mouse IgGFc*HRP. As in preliminary studies, C436A and C438A were the onlyantibodies able to bind cCLB8 that was pre-bound to HuIL-6. FIG. 10illustrates the binding abilities of the 7 anti-id antibodies for cCLB8that is pre-bound to HuIL-6.

In summary, seven monoclonal anti-idiotypic antibodies were producedfrom fusion of murine myeloma cells and spleen cells from a Balb/c mouseimmunized with chimeric anti-Human IL-6 antibody (cCLB8). Five of theanti-id antibodies (C434A, C435A, C436A, C437A, and C439A) were able toblock cCLB8 binding to HuIL-6. Two antibodies (C436A and C438A)possessed the ability to bind cCLB8 that was pre-bound to HuIL-6, andtwo antibodies (C434A and C436A) were virtually unaffected from bindingcCLB8 by any concentration of NHS tested. The broad binding profiles ofthese cCLB8 anti-idiotypic antibodies make some of them potentialcandidates for use as pharmacokinetic probes in serum detection andimmunohistochemical assays.

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

1.-41. (canceled)
 42. A method for treating a lymphoproliferativedisorder or disease in a cell, tissue, organ or animal, comprising;contacting or administering an effective amount of at least one IL-6antibody or specified portion or variant comprising the heavy chain andlight chain complementarity determining regions (CDRs) having the aminoacid sequences of SEQ ID NOS:1-6, and a constant region derived from oneor more human antibodies with, or to, said cell, tissue, organ oranimal.